Methods of treating bladder cancer

ABSTRACT

The present invention provides methods for treating an individual having bladder cancer comprising intravesically administering to the individual an oncolytic virus. Also provided are pharmaceutical compositions and kits for treating bladder cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/713,975, filed on Apr. 5, 2022, which is a continuation of U.S.application Ser. No. 16/605,066, now U.S. Pat. No. 11,338,003, whichadopts the international filing date of Apr. 13, 2018, which is a U.S.national phase application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2018/027549, filed on Apr. 13, 2018, which claimspriority to U.S. Provisional Application No. 62/485,805, filed on Apr.14, 2017, and U.S. Provisional Application No. 62/500,729, filed on May3, 2017, all of which are hereby incorporated by reference in theirentireties.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 744442000402SEQLIST.TXT,date recorded: Jun. 14, 2022, size: 2,396 bytes).

FIELD OF THE INVENTION

The present invention relates to methods of treating bladder carcinomain situ using an oncolytic virus, such as CG0070.

BACKGROUND OF THE INVENTION

Approximately 77,000 new cases of urinary bladder cancer were diagnosedin 2016. Non-muscle invasive bladder cancer (stages Ta, T1, or Carcinomain situ) accounts for 70-80% of these cases while muscle invasivedisease (stage T2 and above) and metastatic disease make up theremaining 20-30%. Bladder cancers are divided into 2 subtypes based ontheir distinct cellular growth patterns: papillary tumors and flattumors. Carcinoma in situ is a flat tumor confined to the surface layerof the bladder. Compared to papillary tumors of the Ta and T1 stage,bladder CIS is more difficult to diagnose, and has a high risk ofprogression to muscle invasive bladder cancer.

Intravesical Bacillus Calmette-Guerin (BCG) is the standard-of-caretreatment for bladder CIS. Patients typically receive an inductioncourse consisting of weekly intravesical installations of BCG for 6weeks, followed by monthly maintenance treatments for 6 to 12 months.However, about 30-40% of patients do not respond to a single course ofBCG treatment. In one study, the overall risk of progression amongBCG-treated CIS patients with a median follow-up of 2.5 years was about14%. See, Witjes, J. A. European urology 45.2 (2004): 142-146. Since thechance of progression in CIS patients failing BCG is significant,cystectomy, including partial and radical cystectomy, remains thetreatment of choice. However, cystectomy is associated with severe sideeffects and adversely affects patients' quality of life. Moreover, therisk of recurrence after radical cystectomy for clinically localizedbladder cancer is high and stage-dependent. See, for example, Bassi P etal. J. Urol. 1999; 161:1494-7. The elderly and patients with renalfailure, who are less tolerable to surgery or chemotherapy, poseadditional clinical challenges for treatment of bladder CIS.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present application provides methods, compositions (includingpharmaceutical compositions) and kits for treating bladder cancer in anindividual comprising intravesical administration of an oncolytic virus,e.g., CG0070.

In one aspect, provided herein is a method of treating bladder cancer inan individual, comprising intravesically administering to the individualan effective amount of an oncolytic virus once per week for three weeksduring a maintenance phase, wherein the oncolytic virus comprises aviral vector comprising a tumor cell-specific promoter operably linkedto a viral gene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule.

In another aspect, provided herein is a method of bladder preservationin an individual comprising intravesically administering to theindividual an effective amount of an oncolytic virus once per week forthree weeks during a maintenance phase, wherein the oncolytic viruscomprises a viral vector comprising a tumor cell-specific promoteroperably linked to a viral gene essential for replication of theoncolytic virus, and a heterologous gene encoding an immune-relatedmolecule.

In some embodiments, according to any of the methods described above,the oncolytic virus is administered once per week for three weeks everysix months during the maintenance phase. In some embodiments, the methodfurther comprises an induction phase prior to the maintenance phase,wherein the induction phase comprises administering to the induvial aneffective amount of oncolytic virus once per week for six weeks. In someembodiments, the start of the induction phase and the start of themaintenance phase are separated by about 3 months. In some embodiments,the start of the induction phase and the start of the maintenance phaseare separated by about 6 months. In some embodiments, the inductionphase comprises administering to the individual an effective amount ofan oncolytic virus once per week for six weeks on month zero and monththree of a treatment regimen.

Also provided herein is a method of treating Ta or T1 bladder cancer inan individual who has not received a transurothelial resection ofbladder tumor (TURBT), comprising intravesically administering to theindividual an effective amount of an oncolytic virus, wherein theoncolytic virus comprises a viral vector comprising a tumorcell-specific promoter operably linked to a viral gene essential forreplication of the oncolytic virus, and a heterologous gene encoding animmune-related molecule.

In one aspect of the present application, there is provided a method oftreating bladder cancer in an individual (such as a human), comprisingintravesically administering to the individual an effective amount of anoncolytic virus, wherein the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the individual only has bladder carcinoma in situ. In someembodiments, the individual does not have a concurrent papillarycarcinoma of Ta or T1 stage. In some embodiments, the individual hasbladder carcinoma in situ and a carcinoma of Ta or Ta stage (CIS+Ta orCIS+T1). In some embodiments, the individual has Ta or T1 bladder cancerbut not carcinoma in situ. In some embodiments, the individual has Ta orT1 bladder cancer and has not received a transurothelial resection ofbladder tumor (TURBT). In some embodiments, the individual hasnon-resectable Ta or T1 stage bladder cancer. In some embodiments, theindividual is unresponsive to BCG treatment. In some embodiments, theindividual has disease reoccurrence subsequent to BCG treatment. In someembodiments, the individual has failed the BCG treatment within about 6months. In some embodiments, the individual is refractory to BCG.

In some embodiments according to any one of the methods described above,the individual has not received a cystectomy. In some embodiments, theindividual has refused or is ineligible for a cystectomy. In someembodiments, the cystectomy is radical cystectomy.

In some embodiments according to any one of the methods described above,the oncolytic virus preferentially replicates in a cancer cell. In someembodiments, the cancer cell is defective in the Rb pathway. In someembodiments, the tumor-specific promoter is an E2F-1 promoter. In someembodiments, the E2F-1 promoter comprises the nucleotide sequence setforth in SEQ ID NO:1.

In some embodiments according to any one of the methods described above,the immune-related molecule is selected from the group consisting ofGM-CSF, IL-2, IL-12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRTO, RIG-I, MDA5, LGP2, andLTαβ. In some embodiments, the immune-related molecule is GM-CSF. Insome embodiments, the heterologous gene is operably linked to a viralpromoter.

In some embodiments according to any one of the methods described above,the oncolytic virus is selected from the group consisting of adenovirus,herpes simplex virus, vaccinia virus, mumps virus, Newcastle diseasevirus, polio virus, measles virus, Seneca valley virus, coxsackie virus,reo virus, vesicular stomatitis virus, maraba and rhabdovirus, andparvovirus. In some embodiments, the oncolytic virus is an oncolyticadenovirus. In some embodiments, the viral gene essential forreplication of the oncolytic virus is selected from the group consistingof E1A, E1B, and E4. In some embodiments, the heterologous gene isoperably linked to an E1 promoter or an E3 promoter. In someembodiments, the oncolytic virus is an adenovirus serotype 5, whereinthe endogenous Ela promoter of a native adenovirus is replaced by thehuman E2F-1 promoter, and the endogenous E3 19kD coding region of thenative adenovirus is replaced by a nucleic acid encoding human GM-CSF.In some embodiments, the oncolytic virus is CG0070.

In some embodiments according to any one of the methods described above,the oncolytic virus is administered at a dose of about 1×10⁸ to about1×10¹⁴ viral particles.

In some embodiments according to any one of the methods described above,the oncolytic virus is administered weekly. In some embodiments, theoncolytic virus is administered for about 1 week to about 6 weeks.

In some embodiments according to any one of the methods described above,the method further comprises intravesically administering to theindividual a transduction enhancing agent prior to the administration ofthe oncolytic virus. In some embodiments, the transduction enhancingagent is N-Dodecyl-β-D-maltoside (DDM).

In some embodiments according to any one of the methods described above,the method is repeated for at least once.

In some embodiments according to any one of the methods described above,the oncolytic virus is not administered in conjunction with an immunecheckpoint modulator.

In some embodiments according to any one of the methods described above,the oncolytic virus is administered as a single therapeutic agent.

Also provided in one aspect is a pharmaceutical composition for treatingbladder cancer in an individual (such as a human), wherein thepharmaceutical composition is intravesically administered to theindividual, and wherein the pharmaceutical composition comprises anoncolytic virus, wherein the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule.

Further provided in one aspect is use of an oncolytic virus in thepreparation of a medicament for treating bladder cancer in an individual(such as a human), wherein the medicament is administered to theindividual intravesically, and wherein the oncolytic virus comprises aviral vector comprising a tumor cell-specific promoter operably linkedto a viral gene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule.

These and other aspects and advantages of the present invention willbecome apparent from the subsequent detailed description and theappended claims. It is to be understood that one, some, or all of theproperties of the various embodiments described herein may be combinedto form other embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of CG0070 and wild type (wt) adenovirustype 5. CG0070 is based on adenovirus serotype 5, but the endogenous Elapromoter and E3 19kD coding region have been replaced by the human E2F-1promoter and a cDNA coding region of human GM-CSF, respectively.

FIG. 2 shows percentage of complete response (CR) in patients havinghigh-grade non-muscle-invasive bladder cancer of various stages at 6months after receiving intravesical CG0070 therapy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for treatingbladder cancer in an individual by intravesically administering to theindividual an effective amount of an oncolytic virus, such as CG0070. Invitro studies have suggested that using a conditionally replicatingoncolytic virus such as CG0070 could be an effective strategy fortreating bladder cancer. RameshN, Ge Y, Ennist DL, Zhu M, Mina M, GaneshS, Reddy PS, Yu DC. CG0070, a conditionally replicating granulocytemacrophage colony-stimulating factor-armed oncolytic adenovirus for thetreatment of bladder cancer. Clin Cancer Res. 2006; 12:305-13. However,the unexpected efficacy of CG0070 in certain patient populations and theeffect of certain dosing regimens as described herein were previouslyunknown. The methods are bladder-preserving, highly efficacious andwell-tolerated in bladder cancer, including those that have previouslyfailed standard-of-care therapies. In a Phase II clinical trial,inventors surprisingly found that 72.2% of patients with pure CIS, whohad previously failed BCG therapy, had complete response (CR) after 6months of intravesical CG0070 therapy. The therapeutic effects of theoncolytic virus can be achieved using the oncolytic virus as amonotherapeutic agent, or without combination with an immune checkpointmodulator. The methods and compositions described herein are especiallyuseful for patients who have high-risk bladder CIS, but refuse toreceive cystectomy, or are ineligible for cystectomy.

Accordingly, one aspect of the present application provides a method oftreating bladder cancer in an individual, comprising intravesicallyadministering to the individual an effective amount of an oncolyticvirus. In some embodiments, the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus is CG0070. In some embodiments, theoncolytic virus is administered as a single therapeutic agent. In someembodiments, the oncolytic virus is not administered in conjunction withan immune checkpoint modulator. In some embodiments, the individual isnot responsive to BCG treatment or has disease reoccurrence subsequentto BCG treatment.

Also provided are compositions (such as pharmaceutical compositions),medicine, kits, and unit dosages useful for the methods describedherein.

I. Definitions

Terms are used herein as generally used in the art, unless otherwisedefined as follows.

As used herein, “bladder carcinoma in situ,” “bladder CIS,” and“carcinoma in situ of the bladder” are used interchangeably to refer tothe clinical stage of bladder cancer characterized by a flat (i.e.,non-papillary) lesion comprising of cytologically malignant cells whichmay involve either full or partial thickness of the urothelium.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: alleviating one ormore symptoms resulting from the bladder cancer, diminishing the extentof the bladder cancer, stabilizing the bladder cancer (e.g., preventingor delaying the worsening of the bladder cancer), preventing or delayingthe spread (e.g., metastasis) of the bladder cancer, preventing ordelaying the recurrence of the bladder cancer, reducing recurrence rateof the bladder cancer, delay or slowing the progression of the bladdercancer, ameliorating the bladder cancer state, providing a remission(partial or total) of the bladder cancer, decreasing the dose of one ormore other medications required to treat the bladder cancer, delayingthe progression of the bladder cancer, increasing the quality of life,and/or prolonging survival. Also encompassed by “treatment” is areduction of pathological consequence of bladder cancer. The methods ofthe invention contemplate any one or more of these aspects of treatment.

“Prior therapy” used herein refers to a therapeutic regime that isdifferent from and was instituted prior to the methods described hereincomprising intravesical administration of the oncolytic virus.

As used herein, an “at risk” individual is a human individual who is atrisk of developing bladder CIS. A human individual “at risk” may or maynot have detectable disease, and may or may not have displayeddetectable disease prior to the treatment methods described herein. “Atrisk” denotes that a human individual has one or more so-called riskfactors, which are measurable parameters that correlate with developmentof muscle invasive bladder cancer, which are described herein. A humanindividual having one or more of these risk factors has a higherprobability of developing bladder CIS than a human individual withoutthese risk factor(s).

“Adjuvant setting” refers to a clinical setting in which an individualhas had a history of bladder cancer, and generally (but not necessarily)been responsive to therapy, which includes, but is not limited to,surgery (e.g., transurethral resection of bladder tumor (“TURBT”),partial cystectomy, or radical cystectomy), radiotherapy, andchemotherapy. Treatment or administration in the “adjuvant setting”refers to a subsequent mode of treatment.

“Neoadjuvant setting” refers to a clinical setting in which the methodis carried out before the primary/definitive therapy.

The term “individual,” “subject,” and “patient” are used interchangeablyherein to describe a mammal, including humans. An individual includes,but is not limited to, human, bovine, horse, feline, canine, rodent, orprimate. In some embodiments, the individual is human. In someembodiments, an individual suffers from bladder CIS. In someembodiments, the individual is in need of treatment.

As used herein, “delaying” the development of bladder cancer means todefer, hinder, slow, retard, stabilize, and/or postpone development ofthe bladder cancer. This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. A method that “delays” development of bladdercancer is a method that reduces probability of disease development in agiven time frame and/or reduces the extent of the bladder cancer in agiven time frame, when compared to not using the method. Suchcomparisons are typically based on clinical studies, using astatistically significant number of subjects. Bladder cancer developmentcan be detectable using standard methods, including, but not limited to,urinary cytology, urethra-cystoscopy (UCS), computed tomography (CTScan, e.g., helical spiral CT scan), endoscopic ultrasound (EUS),endoscopic retrograde cholangiopancreatography (ERCP), laparoscopy, orbiopsy (e.g., percutaneous needle biopsy or fine needle aspiration).Development may also refer to bladder cancer progression that may beinitially undetectable and includes recurrence.

As used herein, by “combination therapy” is meant that a first agent beadministered in conjunction with another agent. “In conjunction with”refers to administration of one treatment modality in addition toanother treatment modality. As such, “in conjunction with” refers toadministration of one treatment modality before, during, or afterdelivery of the other treatment modality to the individual.

As used herein, “monotherapy” refers to administration of a singletherapeutic agent, such as the oncolytic virus, which is not inconjunction with another treatment modality, such as an immunecheckpoint modulator. A monotherapy with the oncolytic virus can beadministered with a pretreatment, such as a transduction enhancing agent(e.g., DDM), which is not considered as a different treatment modalityfor the purpose of this invention.

The term “effective amount” used herein refers to an amount of an agent(such as the oncolytic virus described herein) or composition sufficientto treat a specified disorder, condition or disease such as ameliorate,palliate, lessen, and/or delay one or more of its symptoms. In referenceto cancer, an effective amount comprises an amount sufficient to cause atumor to shrink and/or to decrease the growth rate of the tumor (such asto suppress tumor growth) or to prevent or delay other unwanted cellproliferation in cancer. In some embodiments, an effective amount is anamount sufficient to delay development of bladder cancer. In someembodiments, an effective amount is an amount sufficient to prevent ordelay recurrence. In some embodiments, an effective amount is an amountsufficient to reduce recurrence rate in the individual. In someembodiments, the effective amount is an amount sufficient to inhibittumor metastasis in the individual. An effective amount can beadministered in one or more administrations. The effective amount of theagent or composition may: (i) reduce the number of cancer cells; (ii)reduce tumor size; (iii) inhibit, retard, slow to some extent andpreferably stop cancer cell infiltration into peripheral organs; (iv)inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; (v) inhibit tumor growth; (vi) prevent occurrence and/orrecurrence of tumor; (vii) delay occurrence and/or recurrence of tumor;(viii) reduce recurrence rate of tumor, and/or (ix) relieve to someextent one or more of the symptoms associated with the cancer. As isunderstood in the art, an “effective amount” may be in one or moredoses, i.e., a single dose or multiple doses may be required to achievethe desired treatment endpoint.

The term “simultaneous administration,” as used herein, means that afirst therapy and second therapy in a combination therapy areadministered at the same time. When the first and second therapies areadministered simultaneously, the first and second therapies may becontained in the same composition (e.g., a composition comprising both afirst and second therapy) or in separate compositions (e.g., a firsttherapy is contained in one composition and a second therapy iscontained in another composition).

As used herein, the term “sequential administration” or “in sequence”means that the first therapy and second therapy in a combination therapyare administered with a time separation, for example, of more than about1 minute, such as more than about any of 5, 10, 15, 20, 30, 40, 50, 60,or more minutes. In some cases, the term “sequential administration”means that the first therapy and second therapy in a combination therapyare administered with a time separation of more than about 1 day, suchas more than about any of 1 day to 1 week, 2 weeks, 3 weeks, 4 weeks, 8weeks, 12 weeks, or more weeks. Either the first therapy or the secondtherapy may be administered first. The first and second therapies arecontained in separate compositions, which may be contained in the sameor different packages or kits.

The term “administered immediately prior to” means that the firsttherapy is administered no more than about 15 minutes, such as no morethan about any of 10, 5 or 1 minutes before administration of the secondtherapy. The term “administered immediately after” means that the firsttherapy is administered no more than about 15 minutes, such as no morethan about any of 15, 10 or 1 minutes after administration of the secondtherapy.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to a patient without causing anysignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

An “adverse event” or “AE” as used herein refers to any untoward medicaloccurrence in an individual receiving a marketed pharmaceutical productor in an individual who is participating on a clinical trial who isreceiving an investigational or non-investigational pharmaceuticalagent. The AE does not necessarily have a causal relationship with theindividual's treatment. Therefore, an AE can be any unfavorable andunintended sign, symptom, or disease temporally associated with the useof a medicinal product, whether or not considered to be related to themedicinal product. An AE includes, but is not limited to: anexacerbation of a pre-existing illness; an increase in frequency orintensity of a pre-existing episodic event or condition; a conditiondetected or diagnosed after study drug administration even though it mayhave been present prior to the start of the study; and continuouslypersistent disease or symptoms that were present at baseline and worsenfollowing the start of the study. An AE generally does not include:medical or surgical procedures (e.g., surgery, endoscopy, toothextraction, or transfusion); however, the condition that leads to theprocedure is an adverse event; pre-existing diseases, conditions, orlaboratory abnormalities present or detected at the start of the studythat do not worsen; hospitalizations or procedures that are done forelective purposes not related to an untoward medical occurrence (e.g.,hospitalizations for cosmetic or elective surgery or social/convenienceadmissions); the disease being studied or signs/symptoms associated withthe disease unless more severe than expected for the individual'scondition; and overdose of study drug without any clinical signs orsymptoms.

A “serious adverse event” or (SAE) as used herein refers to any untowardmedical occurrence at any dose including, but not limited to, that: a)is fatal; b) is life-threatening (defined as an immediate risk of deathfrom the event as it occurred); c) results in persistent or significantdisability or incapacity; d) requires in-patient hospitalization orprolongs an existing hospitalization (exception: Hospitalization forelective treatment of a pre-existing condition that did not worsenduring the study is not considered an adverse event. Complications thatoccur during hospitalization are AEs and if a complication prolongshospitalization, then the event is serious); e) is a congenitalanomaly/birth defect in the offspring of an individual who receivedmedication; or f) conditions not included in the above definitions thatmay jeopardize the individual or may require intervention to prevent oneof the outcomes listed above unless clearly related to the individual'sunderlying disease. “Lack of efficacy” (progressive disease) is notconsidered an AE or SAE. The signs and symptoms or clinical sequelaeresulting from lack of efficacy should be reported if they fulfill theAE or SAE definitions.

The following definitions may be used to evaluate response based ontarget lesions: “complete response” or “CR” refers to disappearance ofall target lesions; “partial response” or “PR” refers to at least a 30%decrease in the sum of the longest diameters (SLD) of target lesions,taking as reference the baseline SLD; “stable disease” or “SD” refers toneither sufficient shrinkage of target lesions to qualify for PR, norsufficient increase to qualify for PD, taking as reference the nadir SLDsince the treatment started; and “progressive disease” or “PD” refers toat least a 20% increase in the SLD of target lesions, taking asreference the nadir SLD recorded since the treatment started, or, thepresence of one or more new lesions.

The following definitions of response assessments may be used toevaluate a non-target lesion: “complete response” or “CR” refers todisappearance of all non-target lesions; “stable disease” or “SD” refersto the persistence of one or more non-target lesions not qualifying forCR or PD; and “progressive disease” or “PD” refers to the “unequivocalprogression” of existing non-target lesion(s) or appearance of one ormore new lesion(s) is considered progressive disease (if PD for theindividual is to be assessed for a time point based solely on theprogression of non-target lesion(s), then additional criteria arerequired to be fulfilled.

“Progression free survival” (PFS) indicates the length of time duringand after treatment that the cancer does not grow. Progression-freesurvival includes the amount of time individuals have experienced acomplete response or a partial response, as well as the amount of timeindividuals have experienced stable disease.

“Cystectomy free survival” (CFS) indicates the length of time during andafter treatment that a cystectomy is not required for the patient asdetermined by the physician.

“Predicting” or “prediction” is used herein to refer to the likelihoodthat an individual is likely to respond either favorably or unfavorablyto a treatment regimen.

As used herein, “at the time of starting treatment” or “baseline” refersto the time period at or prior to the first exposure to the treatment.

As used herein, “sample” refers to a composition which contains amolecule which is to be characterized and/or identified, for example,based on physical, biochemical, chemical, physiological, and/or geneticcharacteristics.

It is understood that embodiments of the invention described hereininclude “consisting” and/or “consisting essentially of” embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein, reference to “not” a value or parameter generally meansand describes “other than” a value or parameter. For example, the methodis not used to treat cancer of type X means the method is used to treatcancer of types other than X.

The term “about X-Y” used herein has the same meaning as “about X toabout Y.”

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

II. Methods of Treating Bladder Cancer

One aspect of the present application relates to treatment of bladdercancer in an individual (such as human individual) by localadministration of a virus, such as an oncolytic virus. In this context,local administration of the oncolytic virus encompasses intravesicaladministration of the oncolytic virus. Any of the methods describedherein may be useful for inhibiting growth of a bladder tumor,inhibiting metastasis of a bladder tumor, prolonging survival (such asdisease-free survival, progression-free survival, or cystectomy-freesurvival) of an individual having bladder cancer, causing diseaseremission in an individual having bladder cancer, preventing diseaseprogression of an individual having bladder cancer, and/or improvingquality of life of an individual having bladder cancer. In someembodiments, the method is bladder-preserving or bladder-sparing. Insome embodiments, the bladder-preserving or bladder-sparing method isuseful for improving the quality of life of the individual.

In some embodiments, provided herein is a method of treating bladdercancer comprising intravesically administering to the individual anoncolytic virus (such as CG0070), wherein the oncolytic virus comprisesa viral vector comprising a tumor cell-specific promoter operably linkedto a viral gene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule, wherein theoncolytic virus is administered once per week for three weeks. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is delivered once per week for6 weeks at month 0 and once per week for 3 weeks at month 6.

In some embodiments, the oncolytic virus is administered during aninduction phase and a maintenance phase. In some embodiments, theinduction period comprises administering an oncolytic virus once perweek for six weeks. In some embodiments, the induction period comprisesadministering the oncolytic virus once per week for six weeks on months0 and 3.

In some embodiments, the maintenance phase comprises administering anoncolytic virus once per week for three weeks, following an inductionphase. In some embodiments, the maintenance phase comprises deliveringthe oncolytic virus once per week for three weeks every six months. Insome embodiments, the maintenance phase comprises administering theoncolytic virus weekly for three weeks every 6 months 2, 3, 4, 5, 6times, or as needed. In some embodiments, the maintenance phasecomprises administering an oncolytic virus weekly for three weeks onmonths 6, 12, and 18. In some embodiments, the maintenance phasecomprises administering an oncolytic virus weekly for three weeks onmonths 3, 6, 12, and 18.

In some embodiments, the dosage schedule can be modified based upon theindividual's response to the oncolytic virus. For example, in someembodiments, an individual is administered an oncolytic virus once perweek for six weeks on month 0 and is reevaluated at month 3. In someembodiments, individuals who have a complete response at month 3 begin amaintenance phase comprising administration of an oncolytic virus onceper week for 6 weeks every 6 months. In some embodiments, individualswho do not have a complete response at 3 months receive a secondinduction dose of oncolytic virus once per week for 6 weeks.

Accordingly, provided herein is a method of treating bladder cancer inan individual comprising administering an oncolytic virus weekly forthree weeks every six months, wherein an oncolytic virus (such asCG0070) is administered intravesically, wherein the oncolytic viruscomprises a viral vector comprising a tumor cell-specific promoteroperably linked to a viral gene essential for replication of theoncolytic virus, and a heterologous gene encoding an immune-relatedmolecule. In some embodiments, the oncolytic virus comprises atumor-selective promoter. In some embodiments, the oncolytic virus isnot administered in conjunction with an immune checkpoint modulator. Insome embodiments, the oncolytic virus is administered as a singletherapeutic agent. In some embodiments, the individual is unresponsiveor has disease reoccurrence subsequent to BCG treatment. In someembodiments, the individual has refused or is ineligible for cystectomy(such as radical cystectomy). In some embodiments, the method furthercomprises administering to the individual a transduction enhancing agent(such as DDM) prior to the administration of the oncolytic virus. Insome embodiments, the oncolytic virus is administered at a dose of about1×10⁸ to about 1×10¹⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, the bladder cancer is CIS, CIS+Ta,CIS+T1, CIS without Ta or T1, Ta, or T1 grade bladder cancer. In someembodiments, the individual has Ta or T1 bladder cancer and has notreceived a transurothelial resection of bladder tumor (TURBT). In someembodiments, the bladder cancer is Ta or T1 non-resectable bladdercancer. In some embodiments, the oncolytic virus is administered onmonths 6, 12, and 18 of a treatment regimen. In some embodiments, theoncolytic virus is administered on months 3, 6, 12, and 18 of atreatment regimen. In some embodiments, the oncolytic virus isadministered weekly for three weeks, every six months, as needed.

In some embodiments, provided herein is a method of treating bladdercancer comprising administering an oncolytic virus once per week for sixweeks during an induction phase and once per week for three weeks duringa maintenance phase wherein the an oncolytic virus (such as CG0070) isadministered intervesically, wherein the oncolytic virus comprises aviral vector comprising a tumor cell-specific promoter operably linkedto a viral gene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy). In some embodiments, the method further comprisesadministering to the individual a transduction enhancing agent (such asDDM) prior to the administration of the oncolytic virus. In someembodiments, the oncolytic virus is administered at a dose of about1×10⁸ to about 1×10⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, the bladder cancer is CIS, CIS+Ta,CIS+T1, CIS without Ta or T1, Ta, or T1 grade bladder cancer. In someembodiments, the bladder cancer is Ta or T1 non-respectable bladdercancer. In some embodiments, the induction phase comprises administeringthe oncolytic virus once per week for six weeks on months 0 and 3. Insome embodiments, the maintenance phase comprises administering theoncolytic virus once per week for three weeks on months 6, 12, and 18.In some embodiments, the maintenance phase comprises administering theoncolytic virus once per week for three weeks on months 3, 6, 12, and18. In some embodiments, the maintenance phase comprises administeringthe oncolytic virus once per week for three weeks every six months asneeded.

The present methods have the advantage of reducing the need forcystectomy in patients and thus may be used in bladder sparing methodsand to treat patients who are ineligible for or refuse cystectomy.Accordingly, in some embodiments, provided herein is bladder sparingmethod comprising in an individual comprising administering an oncolyticvirus weekly for three weeks every six months, wherein an oncolyticvirus (such as CG0070) is administered intervesically, wherein theoncolytic virus comprises a viral vector comprising a tumorcell-specific promoter operably linked to a viral gene essential forreplication of the oncolytic virus, and a heterologous gene encoding animmune-related molecule. In some embodiments, the oncolytic viruscomprises a tumor-selective promoter. In some embodiments, the oncolyticvirus is not administered in conjunction with an immune checkpointmodulator. In some embodiments, the oncolytic virus is administered as asingle therapeutic agent. In some embodiments, the individual isunresponsive or has disease reoccurrence subsequent to BCG treatment. Insome embodiments, the individual has refused or is ineligible forcystectomy (such as radical cystectomy). In some embodiments, the methodfurther comprises administering to the individual a transductionenhancing agent (such as DDM) prior to the administration of theoncolytic virus. In some embodiments, the oncolytic virus isadministered at a dose of about 1×10⁸ to about 1×10¹⁴ viral particles(such as about 1×10¹² viral particles). In some embodiments, the bladdercancer is CIS, CIS+Ta, CIS+T1, CIS without Ta or T1, Ta, or T1 gradebladder cancer. In some embodiments, the individual has Ta or T1 bladdercancer and has not received a transurothelial resection of bladder tumor(TURBT). In some embodiments, the bladder cancer is Ta or T1non-resectable bladder cancer. In some embodiments, the oncolytic virusis administered on months 6, 12, and 18 of a treatment regimen. In someembodiments, the oncolytic virus is administered on months 3, 6, 12, and18 of a treatment regimen. In some embodiments, the oncolytic virus isadministered weekly for three weeks, every six months, as needed.

In some embodiments, provided herein is a method of maintenance therapycomprising administering an oncolytic virus weekly for three weeks everysix months, wherein an oncolytic virus (such as CG0070) is administeredintervesically, wherein the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy). In some embodiments, the method further comprisesadministering to the individual a transduction enhancing agent (such asDDM) prior to the administration of the oncolytic virus. In someembodiments, the oncolytic virus is administered at a dose of about1×10⁸ to about 1×10¹⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, the bladder cancer is CIS, CIS+Ta,CIS+T1, CIS without Ta or T1, Ta, or T1 grade bladder cancer. In someembodiments, the individual has Ta or T1 bladder cancer and has notreceived a transurothelial resection of bladder tumor (TURBT). In someembodiments, the bladder cancer is Ta or T1 non-resectable bladdercancer. In some embodiments, the oneolytic virus is administered onmonths 6, 12, and 18 of a treatment regimen. In some embodiments, theoncolytic virus is administered on months 3, 6, 12, and 18 of atreatment regimen. In some embodiments, the oncolytic virus isadministered weekly for three weeks, every six months, as needed.

In some embodiments, the individual receives no more than 21 doses of anoncolytic virus. In some embodiments, the individual receives no morethan 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 doses of anoncolytic virus.

In some embodiments, there is provided a method of treating bladdercancer (such as CIS bladder cancer or Ta or T1 grade bladder cancerwithout TURBT) in an individual, comprising intravesically administeringto the individual an effective amount of an oncolytic virus (such asoncolytic adenovirus) comprising a viral vector comprising a tumorcell-specific promoter operably linked to a viral gene essential forreplication of the oncolytic virus. In some embodiments, the oncolyticvirus comprises a tumor-selective promoter. In some embodiments, thetumor selective promoter allows preferential replication of theoncolytic virus in tumor cells. In some embodiments, the oncolytic viruspreferentially replicates in a cancer cell, such as a cancer celldefective in the Rb pathway. In some embodiments, the oncolytic virus isselected from the group consisting of adenovirus, herpes simplex virus,vaccinia virus, mumps virus, Newcastle disease virus, polio virus,measles virus, Seneca valley virus, coxsackie virus, reo virus,vesicular stomatitis virus, maraba and rhabdovirus, and parvovirus. Insome embodiments, the tumor-specific promoter is an E2F-1 promoter, suchas a human E2F-1 promoter or an E2F-1 promoter comprising the nucleotidesequence set forth in SEQ ID NO:1. In some embodiments, the viral geneessential for replication of the oncolytic virus is selected from thegroup consisting of E1A, E1B, and E4.

In some embodiments, the oncolytic virus further comprises animmune-related molecule (such as cytokine, chemokine, or PRRago (i.e.,pathogen recognition receptor agonist)). In some embodiments, theimmune-related molecule is not an immune checkpoint modulator. In someembodiments, the immune-related molecule is selected from the groupconsisting of GM-CSF, IL-2, IL-12, interferon (such as Type 1, Type 2 orType 3 interferon, e.g., interferon γ), CCL4, CCL19, CCL21, CXCL13,TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I,MDA5, LGP2, and LTαβ. In some embodiments, the immune-related moleculeis selected from the group consisting of STING (i.e., stimulator ofinterferon genes) activators (such as CDN, i.e., cyclic dinucleotides),PRRago (such as CpG, Imiquimod, or Poly I:C), TLR stimulators (such asGS-9620, AED-1419, CYT-003-QbG10, AVE-0675, or PF-7909), and RLRstimulators (such as RIG-I, Mda5, or LGP2 stimulators). In someembodiments, the immune-related molecule induces dendritic cells, Tcells, B cells, and/or T follicular helper cells.

In some embodiments, the immune-related molecule is expressed by theoncolytic virus. For example, the oncolytic virus may comprise a nucleicacid encoding the immune-related molecule, and the nucleic acid can bein the viral vector or on a separate vector. In some embodiments, theoncolytic virus is a virus comprising a viral vector, and wherein theviral vector comprises the nucleic acid encoding the immune-relatedmolecule. In some embodiments, the nucleic acid encoding theimmune-related molecule is operably linked to a viral promoter, such asan E1 promoter, or an E3 promoter.

In some embodiments, the immune-related molecule enhances an immuneresponse in the individual. Immune-related molecules may include, butare not limited to, a cytokine, a chemokine, a stem cell growth factor,a lymphotoxin, an hematopoietic factor, a colony stimulating factor(CSF), erythropoietin, thrombopoietin, tumor necrosis factor-alpha(TNF), TNF-beta, granulocyte-colony stimulating factor (G-CSF),granulocyte macrophage-colony stimulating factor (GM-CSF),interferon-alpha, interferon-beta, interferon-gamma, interferon-lambda,stem cell growth factor designated “S1 factor”, human growth hormone,N-methionyl human growth hormone, bovine growth hormone, parathyroidhormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,NGF-beta, platelet-growth factor, TGF-alpha, TGF-beta, insulin-likegrowth factor-I, insulin-like growth factor-II, macrophage-CSF (M-CSF),IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25,LIF, FLT-3, angiostatin, thrombospondin, endostatin, lymphotoxin,thalidomide, lenalidomide, or pomalidomide.

Thus, in some embodiments, there is provided a method of treatingbladder cancer (such as CIS bladder cancer or Ta or T1 grade bladdercancer without TURBT) in an individual, comprising intravesicallyadministering to the individual an effective amount of an oncolyticvirus, wherein the oncolytic virus comprises a viral vector comprising atumor cell-specific promoter operably linked to a viral gene essentialfor replication of the oncolytic virus and a nucleic acid encodinganimmune-related molecule (such as cytokine or chemokine) operablylinked to a viral promoter. In some embodiments, the oncolytic viruscomprises a tumor-selective promoter. In some embodiments, thetumor-specific promoter is an E2F-1 promoter, such as a human E2F-1promoter or an E2F-1 promoter comprising the nucleotide sequence setforth in SEQ ID NO:1. In some embodiments, the viral gene essential forreplication of the oncolytic virus is selected from the group consistingof E1A, E1B, and E4. In some embodiments, the viral promoter operablylinked to the nucleic acid encoding the immune-related molecule is theE3 promoter. In some embodiments, the immune-related molecule is GM-CSF.In some embodiments, the method further comprises administering to theindividual a transduction enhancing agent (such as DDM) prior to theadministration of the oncolytic virus. In some embodiments, theoncolytic virus is administered at a dose of about 1×10⁸ to about 1×10¹⁴viral particles (such as about 1×10¹² viral particles). In someembodiments, the oncolytic virus is administered weekly. In someembodiments, the oncolytic virus is administered for about 1 week toabout 6 weeks (such as about 6 weeks or about 3 weeks). In someembodiments, the bladder cancer is CIS, CIS+Ta, CIS+T1, Ta, or T1 gradebladder cancer.

In some embodiments, the oncolytic virus is an adenovirus serotype 5. Insome embodiments, the endogenous Ela promoter and E3 19kD coding regionof a native adenovirus is replaced by the human E2F-1 promoter and anucleic acid encoding human GM-CSF. In some embodiments, apolyadenylation signal (PA) is inserted 5′ of the E2F-1 promoter. Insome embodiments, the nucleic acid encoding human GM-CSF is operablylinked to the E3 promoter. In some embodiments, the vector backbone ofthe adenovirus serotype 5 further comprises E2, E4, late protein regionsor inverted terminal repeats (ITRs) identical to the wildtype adenovirusserotype 5 genome. In some embodiments, the oncolytic virus has thegenomic structure as shown in FIG. 1 . In some embodiments, theoncolytic virus is conditionally replicating. In some embodiments, theoncolytic virus preferentially replicates in cancer cells. In someembodiments, the cancer cells are Rb pathway-defective cancer cells. Insome embodiments, the oncolytic virus is CG0070.

Thus, in some embodiments, there is provided a method of treatingbladder cancer (such as CIS bladder cancer or Ta or T1 grade bladdercancer without TURBT) in an individual, comprising intravesicallyadministering to the individual an effective amount of an adenovirusserotype 5, wherein the endogenous Ela promoter and E3 19kD codingregion of a native adenovirus is replaced by the human E2F-1 promoterand a nucleic acid encoding an immune-related molecule (such as cytokineor chemokine, for example, GM-CSF). In some embodiments, thetumor-specific promoter is a human E2F-1 promoter or an E2F-1 promotercomprising the nucleotide sequence set forth in SEQ ID NO:1. In someembodiments, the method further comprises administering to theindividual a transduction enhancing agent (such as DDM) prior to theadministration of the adenovirus. In some embodiments, the adenovirus isadministered at a dose of about 1×10⁸ to about 1×10⁴ viral particles(such as about 1×10¹² viral particles). In some embodiments, theadenovirus is administered weekly. In some embodiments, the adenovirusis administered for about 1 week to about 6 weeks (such as about 6 weeksor about 3 weeks). In some embodiments, the bladder cancer is CIS,CIS+Ta, CIS+T1, Ta, or T1 grade bladder cancer.

In some embodiments, there is provided a method of treating bladdercancer (such as CIS bladder cancer or Ta or T1 grade bladder cancerwithout TURBT), comprising intravesically administering to theindividual an effective amount of CG0070. In some embodiments, themethod further comprises administering to the individual a transductionenhancing agent (such as DDM) prior to the administration of CG0070. Insome embodiments, CG0070 is administered at a dose of about 1×10⁸ toabout 1×10¹⁴ viral particles (such as about 1×10¹² viral particles). Insome embodiments, CG0070 is administered weekly. In some embodiments,CG0070 is administered for about 1 week to about 6 weeks (such as about6 weeks or about 3 weeks). In some embodiments, the bladder cancer isCIS, CIS+Ta, CIS+T1, Ta, or T1 grade bladder cancer.

In some embodiments, the oncolytic virus is not administered to theindividual in conjunction with an immune checkpoint modulator. In someembodiments, the oncolytic virus is administered to the individual as asingle therapeutic agent. As used herein, “immune checkpoint modulator”refers to a molecule or an agent (such as antibody) that inhibits aninhibitory immune checkpoint molecule or an immune-stimulating agentthat activates an immune stimulatory molecule on an immune cell (such asT cell) or a tumor cell. “Immune checkpoint molecules” include moleculesthat turn up an immune signal (i.e., “immune stimulatory molecules”), ormolecules that turn down an immune signal (i.e., “inhibitory immunecheckpoint molecules”) against a tumor cell.

In some embodiments, the oncolytic virus is not administered to theindividual in conjunction with an immune-stimulating agent. Theimmune-stimulating agent may be a natural or engineered ligand of animmune stimulatory molecule selected from the group consisting ofligands of OX40 (e.g., OX40L), ligands of CD-28 (e.g., CD80, CD86),ligands of ICOS (e.g., B7RP1), ligands of 4-1BB (e.g., 4-1BBL,ULTRA4-1BBL™), ligands of CD27 (e.g., CD70), ligands of CD40 (e.g.,CD40L), and ligands of TCR (e.g., MHC class I or class II molecules,IMCgp100). The immune-stimulating agent may also be an antibody selectedfrom the group consisting of anti-CD28 (e.g., TGN-1412), anti-OX40(e.g., MEDI6469, MEDI-0562), anti-ICOS (e.g., MEDI-570), anti-GITR(e.g., TRX518, INBRX-110, NOV-120301), anti-41-BB (e.g., BMS-663513,PF-05082566), anti-CD27 (e.g., BION-1402, Varlilumab and hCD27.15),anti-CD40 (e.g., CP870,893, BI-655064, BMS-986090, APX005, APX005M),anti-CD3 (e.g., blinatumomab, muromonab), and anti-HVEM.

In some embodiments, the oncolytic virus is not administered to theindividual in conjunction with an immune checkpoint inhibitor. Theimmune-checkpoint inhibitor may be a natural or engineered ligand of aninhibitory immune checkpoint molecule selected from the group consistingof ligands of CTLA-4 (e.g., B7.1, B7.2), ligands of TIM3 (e.g.,Galectin-9), ligands of A2a Receptor (e.g., adenosine, Regadenoson),ligands of LAG3 (e.g., IHC class I or MHC class II molecules), ligandsof BTLA (e.g., HVEM, B7-H4), ligands of KIR (e.g., MHC class I or MHCclass II molecules), ligands of PD-1 (e.g., PD-L1, PD-L2), ligands ofIDO (e.g., NKTR-218, Indoximod, NLG919), and ligands of CD47 (e.g.,SIRP-alpha receptor). The immune checkpoint inhibitor may also be anantibody that targets an inhibitory immune checkpoint protein selectedfrom the group consisting of anti-CTLA-4 (e.g., Ipilimumab,Tremelimumab, KAHR-102), anti-TIM3 (e.g., F38-2E2, ENUM005), anti-LAG3(e.g., BMS-986016, IMP701, IP321, C9B7W), anti-KIR (e.g., Lirilumab andIPH2101), anti-PD-1 (e.g., Nivolumab, Pidilizumab, Pembrolizumab,BMS-936559, atezolizumab, Lambrolizumab, MK-3475, AMP-224, AMP-514,STI-All10, TSR-042), anti-PD-LI (e.g., KY-1003 (EP20120194977),MCLA-145, RG7446, BMS-936559, MEDI-4736, MSB0010718C, AUR-012,STI-A1010, PCT/US2001/020964, MPDL3280A, AMP-224, Dapirolizumab pegol(CDP-7657), MEDI-4920), anti-CD73 (e.g., AR-42(OSU-HDAC42,HDAC-42,AR42,AR 42,OSU-HDAC 42,OSU-HDAC-42,NSCD736012,HDAC-42,HDAC 42,HDAC42,NSCD736012,NSC-D736012), MEDI-9447),anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD47 (e.g., CC-90002,TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2aR, anti-B7-1,anti-B7-H4, anti-CD52 (such as alemtuzumab), anti-IL-10, anti-IL-35, andanti-TGF-β (such as Fresolumimab).

Thus, in some embodiments, there is provided a method of treatingbladder cancer (such as CIS bladder cancer or Ta or T1 grade bladdercancer without TURBT) in an individual, comprising intravesicallyadministering to the individual an effective amount of an oncolyticvirus (such as oncolytic adenovirus) comprising a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus and a nucleic acidencoding an immune-related molecule (such as cytokine or chemokine)operably linked to a viral promoter, wherein the oncolytic virus isadministered as a single therapeutic agent. In some embodiments, theoncolytic virus comprises a tumor-selective promoter. In someembodiments, there is provided a method of treating bladder cancer in anindividual, comprising intravesically administering to the individual aneffective amount of an oncolytic virus (such as oncolytic adenovirus)comprising a viral vector comprising a tumor cell-specific promoteroperably linked to a viral gene essential for replication of theoncolytic virus and a nucleic acid encoding animmune-related molecule(such as cytokine or chemokine) operably linked to a viral promoter,wherein the oncolytic virus is not administered in conjunction with animmune checkpoint modulator. In some embodiments, the tumor-specificpromoter is an E2F-1 promoter, such as a human E2F-1 promoter or anE2F-1 promoter comprising the nucleotide sequence set forth in SEQ IDNO:1. In some embodiments, the viral gene essential for replication ofthe oncolytic virus is selected from the group consisting of E1A, E1B,and E4. In some embodiments, the viral promoter operably linked to thenucleic acid encoding the immune-related molecule is the E3 promoter. Insome embodiments, the immune-related molecule is GM-CSF. In someembodiments, the method further comprises administering to theindividual a transduction enhancing agent (such as DDM) prior to theadministration of the oncolytic virus. In some embodiments, theoncolytic virus is administered at a dose of about 1×10⁸ to about 1×10¹⁴viral particles (such as about 1×10¹² viral particles). In someembodiments, the oncolytic virus is administered weekly. In someembodiments, the oncolytic virus is administered for about 1 week toabout 6 weeks (such as about 6 weeks or about 3 weeks). In someembodiments, the bladder cancer is CIS, CIS+Ta, CIS+T1, Ta, or T1 gradebladder cancer.

In some embodiments, there is provided a method of treating bladdercancer (such as CIS bladder cancer or Ta or T1 grade bladder cancerwithout TURBT) in an individual, comprising intravesically administeringto the individual an effective amount of an adenovirus serotype 5,wherein the endogenous Ela promoter and E3 19kD coding region of anative adenovirus is replaced by the human E2F-1 promoter and a nucleicacid encoding an immune-related molecule (such as cytokine or chemokine,for example, GM-CSF), wherein the adenovirus is administered as a singletherapeutic agent. In some embodiments, there is provided a method oftreating bladder carcinoma in situ in an individual, comprisingintravesically administering to the individual an effective amount of anadenovirus serotype 5, wherein the endogenous Ela promoter and E3 19kDcoding region of a native adenovirus is replaced by the human E2F-1promoter and a nucleic acid encoding an immune-related molecule (such ascytokine or chemokine, for example, GM-CSF), wherein the adenovirus isnot administered in conjunction with an immune checkpoint modulator. Insome embodiments, the tumor-specific promoter is a human E2F-1 promoteror an E2F-1 promoter comprising the nucleotide sequence set forth in SEQID NO:1. In some embodiments, the method further comprises administeringto the individual a transduction enhancing agent (such as DDM) prior tothe administration of the adenovirus. In some embodiments, theadenovirus is administered at a dose of about 1×10⁸ to about 1×10¹⁴viral particles (such as about 1×10¹² viral particles). In someembodiments, the adenovirus is administered weekly. In some embodiments,the adenovirus is administered for about 1 week to about 6 weeks (suchas about 3 weeks or about 6 weeks). In some embodiments, the bladdercancer is CIS, CIS+Ta, CIS+T1, Ta, or T1 grade bladder cancer.

In some embodiments, there is provided a method of treating bladdercancer (such as CIS bladder cancer or Ta or T1 grade bladder cancerwithout TURBT) in an individual, comprising intravesically administeringto the individual an effective amount of CG0070, wherein CG0070 isadministered as a single therapeutic agent. In some embodiments, thereis provided a method of treating bladder carcinoma in situ in anindividual, comprising intravesically administering to the individual aneffective amount of CG0070, wherein CG0070 is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the method further comprises administering to the individual atransduction enhancing agent (such as DDM) prior to the administrationof CG0070. In some embodiments, CG0070 is administered at a dose ofabout 1×10⁸ to about 1×10¹⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, CG0070 is administered weekly. In someembodiments, CG0070 is administered for about 1 week to about 6 weeks(such as about 3 weeks or about 6 weeks). In some embodiments, thebladder cancer is CIS, CIS+Ta, CIS+T1, Ta, or T1 grade bladder cancer.

Patient Populations

The methods described herein can be used to treat a variety of bladdercancers. In some embodiments, the individual has a bladder cancer at astage Ta, T1, or CIS (TIS) as determined according to the TNM stagingsystem by the American Joint Committee on Cancer (AJCC) guidelines.

In some embodiments the bladder cancer is CIS. In some embodiments, thebladder CIS is transitional cell carcinoma or urothelial carcinoma. Insome embodiments, the bladder CIS is metastatic urothelial carcinoma. Insome embodiments, the bladder CIS is located in the urothelium of thebladder. In some embodiments, the bladder CIS is located in the upperurinary tract. In some embodiments, the bladder CIS is in the ureter. Insome embodiments, the bladder CIS is in the urethra. In someembodiments, the bladder CIS is in the renal pelvis.

“Bladder carcinoma in situ” is a high-grade bladder cancer, also knownas Stage 0 is, Tis, flat, or bladder CIS. Bladder CIS is found only onthe inner lining of the bladder, and it has not grown in toward thehollow part of the bladder, and it has not spread to the thick layer ofmuscle or connective tissue of the bladder. Bladder CIS is considered anaggressive disease, and believed to be a precursor of invasive bladdercancer. According to NCCN Guidelines for Bladder Cancer, currentstandard therapy for bladder CIS is resection followed by intraveiscaltherapy with BCG. BCG is generally given once a week for 6 weeks,followed by a rest period of 4 to 6 weeks, with a full re-evaluation atweek 12 after the start of therapy. If the patient is unable to tolerateBCG, intravesical mitomycin C may be administered.

In some embodiments, the individual has a pathological stage of “pureCIS” or “CIS,” wherein the individual has one or more bladder carcinomasin situ only. In some embodiments, the individual has a pathologicalstage of Tis, N0, M0. In some embodiments, the bladder CIS is a primarytumor. In some embodiments, the bladder CIS is a recurrent tumor. Insome embodiments, the individual has at least about any one of 1, 2, 3,4, 5, 10, or more bladder carcinomas in situ.

In some embodiments, the individual has concurrently one or more bladdercarcinomas in situ and one or more papillary carcinomas of Ta or T1stage. In some embodiments, the individual has a pathological stage of“CIS+Ta,” wherein the individual has concurrently one or more bladdercarcinomas in situ and one or more Ta stage papillary carcinomas. Insome embodiments, the individual has a pathological stage of “CIS+T1,”wherein the individual has concurrently one or more bladder carcinomasin situ and one or more T1 stage papillary carcinomas.

“Papillary carcinoma” refers to bladder tumors that grow in slender,finger-like projections from the inner surface of the bladder toward thehollow center. Papillary tumors often grow toward the center of thebladder without growing into the deeper bladder layers. These tumors arecalled non-invasive papillary cancers. Very low-grade (slow growing),non-invasive papillary cancer is sometimes called papillary urothelialneoplasm of low-malignant potential (PUNLMP).

“Ta” stage papillary carcinoma refers to papillary carcinoma that isfound on the surface of the inner lining of the bladder. Cancer cellsare grouped together and can often be easily removed. The cancer has notinvaded the muscle or connective tissue of the bladder wall. Tapapillary carcinoma is also referred to as noninvasive papillaryurothelial carcinoma, or Stage 0a bladder cancer.

“T1” stage papillary carcinoma refers to papillary carcinoma that hasgrown through the inner lining of the bladder into the lamina propria.It has not spread to the thick layer of muscle in the bladder wall or tolymph nodes or other organs. In some embodiments, the carcinoma hasinvaded subepithelial connective tissue.

In some embodiments, the individual does not have a concurrent papillarycarcinoma with CIS. In some embodiments, the individual does not havelow-grade or high-grade papillary carcinoma. In some embodiments, theindividual does not have high-grade papillary carcinoma. In someembodiments, the individual does not have a concurrent papillarycarcinoma of low-grade Ta stage. In some embodiments, the individualdoes not have a concurrent papillary carcinoma of high-grade Ta stage.In some embodiments, the individual does not have a concurrent papillarycarcinoma of low-grade T1 stage. In some embodiments, the individualdoes not have a concurrent papillary carcinoma of high-grade T1 stage.In some embodiments, the individual does not have a concurrent papillarycarcinoma of Ta or T1 stage.

In some embodiments, the individual has a papillary carcinoma. In someembodiments, the individual has a low-grade or high-grade papillarycarcinoma concurrent with CIS. In some embodiments, the individual has ahigh-grade papillary carcinoma. In some embodiments, the individual hasa concurrent papillary carcinoma of low-grade Ta stage. In someembodiments, the individual has a concurrent papillary carcinoma ofhigh-grade Ta stage. In some embodiments, the individual does not have aconcurrent papillary carcinoma of low-grade T1 stage. In someembodiments, the individual does not have a concurrent papillarycarcinoma of high-grade T1 stage. In some embodiments, the individualdoes not have a concurrent papillary carcinoma of Ta or T1 stage.

In some embodiments, the individual has a papillary carcinoma withoutCIS. In some embodiments, the individual has a low-grade or high-gradepapillary carcinoma. In some embodiments, the individual has ahigh-grade papillary carcinoma. In some embodiments, the individual hasa concurrent papillary carcinoma of low-grade Ta stage. In someembodiments, the individual has a concurrent papillary carcinoma ofhigh-grade Ta stage. In some embodiments, the individual does not have aconcurrent papillary carcinoma of low-grade T1 stage. In someembodiments, the individual does not have a concurrent papillarycarcinoma of high-grade T1 stage. In some embodiments, the individualhas Ta or T1 stage bladder cancer without CIS.

Bladder cancer can be detected and staged using any known methods in theart, including, but not limited to, urinary cytology, urethra-cystoscopy(UCS), biopsy (such as transurethral resection of bladder tumor, or“TURBT”), computed tomography (CT or CAT) scan (e.g., CT urography),magnetic resonance imaging (MRI, i.e., MR urography), positron emissiontomography (PET or PET-CT) scan, ultrasound (e.g., renal ultrasound),ureteroscopy, and x-ray imaging (e.g., chest imaging). A distinctfeature of bladder CIS is the fact that coherence and adherence of CIScells are decreased. As a result, more cells are present in urine, whichcan be detected by urinary cytology. Additionally, bladder CIS isassociated with an increased chance of denuded epithelium upon biopsy.Tumor cells from urinary samples or biopsy samples can be examined usingimmunohistochemistry, ELISA, RT-PCR, or other suitable methods to detecturinary markers for bladder CIS or papillary tumors. Fluorescencecystoscopy methods, e.g., those using a porphyrin-based phososensitizer,may be especially suitable for detecting CIS lesions.

Thus, in some embodiments, there is provided a method of treating anindividual having bladder carcinoma in situ only, comprisingintravesically administering to the individual an oncolytic virus (suchas CG0070), wherein the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the method further comprises administering to theindividual a transduction enhancing agent (such as DDM) prior to theadministration of the oncolytic virus. In some embodiments, theoncolytic virus is administered at a dose of about 1×10⁸ to about 1×10¹⁴viral particles (such as about 1×10¹² viral particles). In someembodiments, the oncolytic virus is administered weekly. In someembodiments, the oncolytic virus is administered for about 1 week toabout 6 weeks (such as about 3 weeks or about 6 weeks).

In some embodiments, there is provided a method of treating anindividual having bladder CIS, comprising intravesically administeringto the individual an oncolytic virus (such as CG0070), wherein theoncolytic virus comprises a viral vector comprising a tumorcell-specific promoter operably linked to a viral gene essential forreplication of the oncolytic virus, and a heterologous gene encoding animmune-related molecule, and wherein the individual does not have aconcurrent papillary carcinoma of Ta or T1 stage. In some embodiments,the oncolytic virus is not administered in conjunction with an immunecheckpoint modulator. In some embodiments, the oncolytic virus isadministered as a single therapeutic agent. In some embodiments, themethod further comprises administering to the individual a transductionenhancing agent (such as DDM) prior to the administration of theoncolytic virus. In some embodiments, the oncolytic virus isadministered at a dose of about 1×10⁸ to about 1×10¹⁴ viral particles(such as about 1×10¹² viral particles). In some embodiments, theoncolytic virus is administered weekly. In some embodiments, theoncolytic virus is administered for about 1 week to about 6 weeks (suchas about 3 weeks or about 6 weeks).

In some embodiments, there is provided a method of treating anindividual having bladder CIS (such as pure CIS), comprisingintravesically administering to the individual CG0070, wherein CG0070 isadministered weekly at a dose of about 1×10⁸ to about 1×10¹⁴ viralparticles (such as about 1×10¹² viral particles) for about 3 or about 6weeks. In some embodiments, the method further comprises administeringto the individual a transduction enhancing agent (such as DDM) prior tothe administration of CG0070. In some embodiments, CG0070 is notadministered in conjunction with an immune checkpoint modulator. In someembodiments, CG0070 is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy).

In some embodiments, there is provided a method of treating anindividual having T1 stage bladder cancer with CIS, comprisingintravesically administering to the individual CG0070, wherein CG0070 isadministered weekly at a dose of about 1×10⁸ to about 1×10¹⁴ viralparticles (such as about 1×10¹² viral particles) for about 3 or about 6weeks. In some embodiments, the method further comprises administeringto the individual a transduction enhancing agent (such as DDM) prior tothe administration of CG0070. In some embodiments, CG0070 is notadministered in conjunction with an immune checkpoint modulator. In someembodiments, CG0070 is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy).

In some embodiments, there is provided a method of treating anindividual having Ta stage bladder cancer with CIS, comprisingintravesically administering to the individual CG0070, wherein CG0070 isadministered weekly at a dose of about 1×10⁸ to about 1×10¹⁴ viralparticles (such as about 1×10¹² viral particles) for about 6 weeks. Insome embodiments, the method further comprises administering to theindividual a transduction enhancing agent (such as DDM) prior to theadministration of CG0070. In some embodiments, CG0070 is notadministered in conjunction with an immune checkpoint modulator. In someembodiments, CG0070 is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy).

In some embodiments, there is provided a method of treating anindividual having T1 stage bladder cancer without CIS, comprisingintravesically administering to the individual CG0070, wherein CG0070 isadministered weekly at a dose of about 1×10⁸ to about 1×10¹⁴ viralparticles (such as about 1×10¹² viral particles) for about 6 weeks. Insome embodiments, the method further comprises administering to theindividual a transduction enhancing agent (such as DDM) prior to theadministration of CG0070. In some embodiments, CG0070 is notadministered in conjunction with an immune checkpoint modulator. In someembodiments, CG0070 is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy).

In some embodiments, there is provided a method of treating anindividual having Ta stage bladder cancer without CIS, comprisingintravesically administering to the individual CG0070, wherein CG0070 isadministered weekly at a dose of about 1×10⁸ to about 1×10¹⁴ viralparticles (such as about 1×10¹² viral particles) for about 6 weeks. Insome embodiments, the method further comprises administering to theindividual a transduction enhancing agent (such as DDM) prior to theadministration of C00070. In some embodiments, CG0070 is notadministered in conjunction with an immune checkpoint modulator. In someembodiments, CG0070 is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy).

In some embodiments, the individual has early stage bladder cancer,non-metastatic bladder cancer, non-invasive bladder cancer,non-muscle-invasive bladder cancer, primary bladder cancer, locallyadvanced bladder cancer (such as unresectable locally advanced bladdercancer), metastatic bladder cancer, or bladder cancer in remission. Insome embodiments, the bladder cancer is localized resectable, localizedunresectable, or unresectable. In some embodiments, the bladder canceris a high grade, non-muscle-invasive cancer that has been refractory tostandard intra-bladder infusion (intravesical) therapy. In someembodiments, the individual has a high grade, non-muscle-invasive CISthat has been refractory to standard intra-bladder infusion(intravesical) therapy. In some embodiments, the individual hasurothelial (i.e., transitional cell) carcinoma.

The methods provided herein can be used to treat an individual (e.g.,human) who has been diagnosed with or is suspected of having bladdercancer. In some embodiments, the individual has undergone a tumorresection, such as TURBT, or partial cystectomy. In some embodiments,the individual has previously received TURBT alone. In some embodiments,the individual has previously received TURBT and concurrentchemoradiotherapy. In some embodiments, the individual has previouslyreceived repeated TURBT. In some embodiments, the individual haspreviously received TURBT for at least about any one of 1, 2, 3, 4, ormore times. In some embodiments, the individual has previously receivedmaximal TURBT. In some embodiments, the individual has not received atumor resection. According to the NCCN Guidelines for Bladder Cancer,bladder preservation with maximal TURBT and concurrent chemoradiotherapyis generally reserved for patients with smaller solitary tumors,negative nodes, no carcinoma in situ, no tumor-related hydronephrosis,and good pre-treatment bladder function.

In some embodiments, the methods provided herein can be used to treat anindividual with bladder cancer who has not received a TURBT, or partialcystectomy prior to treatment with C60070. In some embodiments, theindividual has a Ta or T1 stage cancer. In some embodiments, theindividual is considered unresectable or with residual disease. In someembodiments, the individual has an underlying medical condition thatdoes not permit adequate resection.

Accordingly, in some embodiments, there is provided a method of treatingan individual having T1 or Ta stage bladder cancer that is has not beenresected or is considered non-resectable, comprising intravesicallyadministering to the individual CG0070, wherein CG0070 is administeredweekly at a dose of about 1×10⁸ to about 1×10¹⁴ viral particles (such asabout 1×10¹² viral particles) for about 6 weeks. In some embodiments,the method further comprises administering to the individual atransduction enhancing agent (such as DDM) prior to the administrationof CG0070. In some embodiments, CG0070 is not administered inconjunction with an immune checkpoint modulator. In some embodiments,CG0070 is administered as a single therapeutic agent. In someembodiments, the individual is unresponsive or has disease reoccurrencesubsequent to BCG treatment. In some embodiments, the individual hasrefused or is ineligible for cystectomy (such as radical cystectomy).

In some embodiments, the individual has refused cystectomy, such aspartial cystectomy or radical cystectomy. In some embodiments, theindividual is ineligible for cystectomy, such as partial cystectomy orradical cystectomy. In some embodiments, the individual is lesstolerable to cystectomy. In some embodiments, the individual ismedically inoperable. In some embodiments, the individual has previouslyreceived a full course of external-beam radiotherapy, and has bulkyresidual disease. In some embodiments, the individual has previouslyreceived a bladder-preserving therapy. In some embodiments, theindividual is an elderly patient. In some embodiments, the individualcannot tolerate cystectomy because of age. In some embodiments, theindividual is at least about any of 60, 65, 70, 75, 80, 85, 90, 95 yearsold or older. In some embodiments, the individual has renal deficiencyor renal failure. According to NCCN Guidelines for Bladder Cancer,partial cystectomy is indicated for T2 muscle invasive disease withsolitary lesion in location amenable to segmental resection withadequate margins, and no carcinoma in situ is determined by randombiopsy. Radical cystectomy or cystoprostatectomy is indicated forresidual high-grade T1, and muscle-invasive disease. Cystectomy isnormally given within 3 months of diagnosis if no therapy is given tothe patient. Additionally, Cystectomy is indicated for recurrent orpersistent bladder cancer at Ta, T1 or CIS stage, after TURBT or BCGtreatment.

In some embodiments, the individual is ineligible for radical cystectomyunder the National Comprehensive Cancer Network (NCCN) guidelines. Forexample, the individual may be unfit for curative therapy due tofrailty. Prior to the present methods, such individuals typicallyreceived palliative radiation without chemotherapy (3.5 Gy/fraction—10treatments; or 7Gy/fraction—7 treatments; TURBT; or no treatment).

In some embodiments, the individual cannot tolerate radical cystectomybased upon the American Society of Anesthesiology (ASA) guidelines. Forexample the individual who cannot tolerate radial cystectomy may bedeemed medically unfit for surgery requiring general or epiduralanesthesia.

In other embodiments, the individual may lack operative post-operativecare infrastructure or personal as determined by the ComprehensiveGeriatric Assessment provided by the American Society ofAnesthesiologists. Under these guidelines, an individual is deemed frailif he or she shows abnormal independent activities of daily living,severe malnutrition, cognitive impairment, or comorbidities cumulativeillness rating scale for geriatrics (CISR-G) grades 3-4.

Thus, in some embodiments, there is provided a method of treating anindividual having bladder cancer, comprising intravesicallyadministering to the individual an oncolytic virus (such as CG0070),wherein the oncolytic virus comprises a viral vector comprising a tumorcell-specific promoter operably linked to a viral gene essential forreplication of the oncolytic virus, and a heterologous gene encoding animmune-related molecule, and wherein the individual has refused or isineligible for cystectomy (such as radical cystectomy). In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the method further comprises administering to theindividual a transduction enhancing agent (such as DDM) prior to theadministration of the oncolytic virus. In some embodiments, theoncolytic virus is administered at a dose of about 1×10⁸ to about 1×10¹⁴viral particles (such as about 1×10¹² viral particles). In someembodiments, the oncolytic virus is administered weekly. In someembodiments, the oncolytic virus is administered for about 1 week toabout 6 weeks (such as about 3 weeks or about 6 weeks).

The methods of the present invention also provide important andsignificant treatment benefits compared to standard therapeutic regimensthat call for removal of the bladder. The present invention also has theadvantage of being useful as a bladder sparing protocol for individualswho are eligible for a cystectomy, but elect not to have a cystectomy.The present methods result in a greatly improved quality of life forindividuals, who may be able to retain their bladder after havingbladder cancer, compared to the presently available treatments.

Accordingly, in some embodiments, there is provided herein is a bladdersparing method comprising intravesically administering to the individualan oncolytic virus (such as CG0070), wherein the oncolytic viruscomprises a viral vector comprising a tumor cell-specific promoteroperably linked to a viral gene essential for replication of theoncolytic virus, and a heterologous gene encoding an immune-relatedmolecule, and wherein the individual has refused or is ineligible forcystectomy (such as radical cystectomy). In some embodiments, theoncolytic virus comprises a tumor-selective promoter. In someembodiments, the oncolytic virus is not administered in conjunction withan immune checkpoint modulator. In some embodiments, the oncolytic virusis administered as a single therapeutic agent. In some embodiments, themethod further comprises administering to the individual a transductionenhancing agent (such as DDM) prior to the administration of theoncolytic virus. In some embodiments, the oncolytic virus isadministered at a dose of about 1×10⁸ to about 1×10¹⁴ viral particles(such as about 1×10¹² viral particles). In some embodiments, theoncolytic virus is administered weekly. In some embodiments, theoncolytic virus is administered for about 1 week to about 6 weeks (suchas about 3 weeks or about 6 weeks).

In some embodiments, the individual has been previously treated forbladder cancer (also referred to as the “prior therapy”). In someembodiments, the prior therapy comprises one or more (such as 1, 2, 3,4, 5, or more) treatment modalities, including, but are not limited tosurgery (such as transurethral resection of bladder tumor, or partialcystectomy), intravesical therapy (such as BCG or intravesicalchemotherapy), radiation therapy, chemotherapy, immunotherapy, andcombinations thereof.

In some embodiments, the present methods are especially suited forindividuals who relapse after BCG therapy after at least 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 months. In some embodiments, the methods areespecially suitable for individuals who relapse at least 9 months afterreceiving BCG therapy. In some embodiments, the individual has CIS.

In some embodiments, the individual has been previously treated with astandard therapy for bladder cancer. In some embodiments, the individualhas been previously treated with a standard therapy for non-invasivepapillary carcinoma. In some embodiments, the individual has beenpreviously treated with a standard therapy for bladder CIS. In someembodiments, the prior standard therapy is an intravesical therapy, suchas intravesical chemotherapy or intravesical immunotherapy. In someembodiments, the prior standard therapy is treatment with mitomycin C.In some embodiments, the prior standard therapy is treatment withinterferon (such as interferon-α). In some embodiments, the priorstandard therapy is treatment with platinum-based agents. In someembodiments, the prior standard therapy is treatment with mitomycin andthiotepa. In some embodiments, the prior standard therapy is treatmentwith cisplatin, doxorubicin, gemcitabine, and valrubicin. In someembodiments, the prior standard therapy is BCG treatment.

In some embodiments, the individual has bladder CIS in remission,progressive bladder CIS, persistent bladder CIS, or recurrent bladderCIS. In some embodiments, the individual is resistant to treatment ofbladder CIS with other agents (such as BCG, or chemotherapy agent). Insome embodiments, the individual is initially responsive to treatment ofbladder CIS with other agents (such as BCG, or chemotherapy agent) buthas progressed after treatment. In some embodiments, the individual haspersistent or recurrent bladder CIS after receiving TURBT. In someembodiments, the individual has persistent or recurrent bladder CISafter receiving an intravesical therapy, such as intravesical BCG orintravesical chemotherapy (e.g., mitomycin C). In some embodiments, theindividual has persistent or recurrent bladder CIS after receiving atleast 2 consecutive cycles or anintravesical therapy, and/or at leasttwo intravesical agents (e.g., intraveiscal BCG followed by intravesicalmitomycin C). In some embodiments, the individual has persistent orrecurrent bladder CIS after receiving a combination of TURBT and one ormore intravesical therapy.

In some embodiments, the individual has recurrent bladder CIS (such aspure CIS) after a prior therapy (such as prior standard therapy, forexample intravesical BCG). For example, the individual may be initiallyresponsive to the treatment with the prior therapy, but develops bladderCIS after about any of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36,48, or 60 months upon the cessation of the prior therapy.

In some embodiments, the individual has previously received BCGtreatment. In some embodiments, the individual has previously receivedat least one (such as at least about any one of 1, 2, 3, 4, 5, 6, ormore) course of intravesical BCG treatment. In some embodiments, theintravesical BCG treatment comprises weekly intravesical instillation ofBCG for at least about 4 weeks, such as at least about any one of 5, 6,7, 8, 9, or more weeks. In some embodiments, the individual haspreviously received an induction course of intravesical BCG treatment(such as about 4 to about 9, e.g., about 6 weekly BCG administration).In some embodiments, the individual has previously received an inductioncourse and at least one maintenance course of intravesical BCGtreatment. In some embodiments, the individual has previously received a6-233k induction course of BCG followed by maintenance with three weeklyinstillations at about any one or more of months 3, 6, 12, 18, 24, 30and 36. In some embodiments, the individual has been on maintenanceintravesical BCG treatment for at least about 3 months, such as at leastabout any one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 monthsor longer. In some embodiments, the individual has received BCGtreatment within about any one of 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or1 month before receiving the viral treatment of the present application.In some embodiments, the individual has severe adverse effects afterintravesical BCG treatment. In some embodiments, the individual hasbacteriuria, persistent gross hematuria, persistent severe localsymptoms, or systemic symptoms. In some embodiments, the individualcannot tolerate the intravesical BCG treatment even after dosereduction.

In some embodiments, the individual has failed the BCG treatment. Insome embodiments, the individual has failed the BCG treatment withinabout any one of 24, 22, 20, 18, 16, 14, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 month after the last administration of BCG. In someembodiments, the individual has failed the induction course ofintravesical BCG treatment.

In some embodiments, the individual has failed the BCG treatment duringor after the maintenance course(s).

In some embodiments, the individual is unresponsive to the BCGtreatment. In some embodiments, the individual had partial response tothe BCG treatment. In some embodiments, the individual could nottolerate the adverse effects of the BCG treatment. In some embodiments,the individual has disease reoccurrence subsequent to the BCG treatment.In some embodiments, the individual has disease reoccurrence after nomore than about any one of 24, 22, 20, 18, 16, 14, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, or 1 month after the last administration of BCG (such asBCG induction or BCG maintenance). In some embodiments, the individualhas disease progression despite receiving maintenance courses of the BCGtreatment. In some embodiments, the individual had bladder CIS prior tothe BCG treatment. In some embodiments, the individual did not havebladder CIS prior to the BCG treatment. In some embodiments, theindividual had papillary tumors (such as stage Ta or T1) prior to theBCG treatment. In some embodiments, the individual had both bladder CISand papillary tumors (such as stage Ta or T1) prior to the BCGtreatment.

Thus, in some embodiments, there is provided a method of treatingBCG-unresponsive non-muscle-invasive bladder cancer (such asBCG-unresponsive bladder CIS) in an individual, comprisingintravesically administering to the individual an oncolytic virus (suchas CG0070), wherein the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the individual only has bladder CIS. In someembodiments, the individual has refused or is ineligible for cystectomy(such as radical cystectomy). In some embodiments, the method furthercomprises administering to the individual a transduction enhancing agent(such as DDM) prior to the administration of the oncolytic virus. Insome embodiments, the oncolytic virus is administered at a dose of about1×10⁸ to about 1×10¹⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, the oncolytic virus is administeredweekly. In some embodiments, the oncolytic virus is administered forabout 1 week to about 6 weeks (such as about 6 weeks).

In some embodiments, there is provided a method of treating anindividual having bladder CIS, comprising intravesically administeringto the individual an oncolytic virus (such as CG0070), wherein theoncolytic virus comprises a viral vector comprising a tumorcell-specific promoter operably linked to a viral gene essential forreplication of the oncolytic virus, and a heterologous gene encoding animmune-related molecule, and wherein the individual has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theoncolytic virus is not administered in conjunction with an immunecheckpoint modulator. In some embodiments, the oncolytic virus comprisesa tumor-selective promoter. In some embodiments, the oncolytic virus isadministered as a single therapeutic agent. In some embodiments, theindividual only has bladder CIS. In some embodiments, the individual hasrefused or is ineligible for cystectomy (such as radical cystectomy). Insome embodiments, the method further comprises administering to theindividual a transduction enhancing agent (such as DDM) prior to theadministration of the oncolytic virus. In some embodiments, theoncolytic virus is administered at a dose of about 1×10⁸ to about 1×10¹⁴viral particles (such as about 1×10¹² viral particles). In someembodiments, the oncolytic virus is administered weekly. In someembodiments, the oncolytic virus is administered for about 1 week toabout 6 weeks (such as about 3 weeks or about 6 weeks).

Endpoints

In some embodiments, the method prevents progression of the bladdercancer in the individual. In some embodiments, the method preventprogression of the non-muscle invasive bladder cancer to muscle invasivebladder cancer by at least about any one of 3, 6, 9, 12, 18, 24, 30, 36,42, 48, 54, 60 or more months.

In some embodiments, there is provided a method of preventing diseaseprogression in an individual having bladder cancer, comprisingintravesically administering to the individual an oncolytic virus (suchas CG0070), wherein the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy). In some embodiments, the method further comprisesadministering to the individual a transduction enhancing agent (such asDDM) prior to the administration of the oncolytic virus. In someembodiments, the oncolytic virus is administered at a dose of about1×10⁸ to about 1×10¹⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, the oncolytic virus is administeredweekly. In some embodiments, the oncolytic virus is administered forabout 1 week to about 6 weeks (such as about 3 weeks or about 6 weeks).In some embodiments, the bladder cancer is CIS, CIS+Ta, CIS+T1, Ta, orT1 grade bladder cancer.

In some embodiments, the method inhibits growth or reduces the size ofthe bladder cancer. In some embodiments, the size of the bladder canceris reduced for at least about 10% (including for example at least aboutany of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%).

In some embodiments, there is provided a method of inhibiting tumorgrowth or reducing tumor size in an individual having bladder cancer,comprising intravesically administering to the individual an oncolyticvirus (such as CG0070), wherein the oncolytic virus comprises a viralvector comprising a tumor cell-specific promoter operably linked to aviral gene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy). In some embodiments, the method further comprisesadministering to the individual a transduction enhancing agent (such asDDM) prior to the administration of the oncolytic virus. In someembodiments, the oncolytic virus is administered at a dose of about1×10⁸ to about 1×10¹⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, the oncolytic virus is administeredweekly. In some embodiments, the oncolytic virus is administered forabout 1 week to about 6 weeks (such as about 3 weeks or about 6 weeks).In some embodiments, the bladder cancer is CIS, CIS+Ta, CIS+T1, Ta, orT1 grade bladder cancer.

In some embodiments, the method causes disease remission (partial orcomplete) in the individual. In some embodiments, the individual hasdisease remission for at least about any one of 2, 3, 4, 5, 6, 12, 24,or more months.

In some embodiments, there is provided a method of causing diseaseremission (such as partial or complete remission) in an individualhaving bladder CIS (such as pure CIS), comprising intravesicallyadministering to the individual an oncolytic virus (such as CG0070),wherein the oncolytic virus comprises a viral vector comprising a tumorcell-specific promoter operably linked to a viral gene essential forreplication of the oncolytic virus, and a heterologous gene encoding animmune-related molecule. In some embodiments, the oncolytic viruscomprises a tumor-selective promoter. In some embodiments, the oncolyticvirus is not administered in conjunction with an immune checkpointmodulator. In some embodiments, the oncolytic virus is administered as asingle therapeutic agent. In some embodiments, the individual isunresponsive or has disease reoccurrence subsequent to BCG treatment. Insome embodiments, the individual has refused or is ineligible forcystectomy (such as radical cystectomy). In some embodiments, the methodfurther comprises administering to the individual a transductionenhancing agent (such as DDM) prior to the administration of theoncolytic virus. In some embodiments, the oncolytic virus isadministered at a dose of about 1×10⁸ to about 1×10¹⁴ viral particles(such as about 1×10¹² viral particles). In some embodiments, theoncolytic virus is administered weekly. In some embodiments, theoncolytic virus is administered for about 1 week to about 6 weeks (suchas about 3 weeks or about 6 weeks).

In some embodiments, the method inhibits tumor metastasis in theindividual. In some embodiments, at least about 10% (including forexample at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or100%) metastasis is inhibited. In some embodiments, a method ofinhibiting metastasis to lymph node is provided. In some embodiments, amethod of inhibiting metastasis to the lung is provided. Metastasis canbe assessed by any known methods in the art, such as by blood tests,bone scans, x-ray scans, CT scans, PET scans, and biopsy.

In some embodiments, there is provided a method of inhibiting tumormetastasis in an individual having bladder cancer, comprisingintravesically administering to the individual an oncolytic virus (suchas CG0070), wherein the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy). In some embodiments, the method further comprisesadministering to the individual a transduction enhancing agent (such asDDM) prior to the administration of the oncolytic virus. In someembodiments, the oncolytic virus is administered at a dose of about1×10⁸ to about 1×10¹⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, the oncolytic virus is administeredweekly. In some embodiments, the oncolytic virus is administered forabout 1 week to about 6 weeks (such as about 3 weeks or about 6 weeks).In some embodiments, the bladder cancer is CIS, CIS+Ta, CIS+T1, Ta, orT1 grade bladder cancer.

In some embodiments, the method prolongs survival (such as disease freesurvival, progression-free survival, or cystectomy-free survival) in theindividual. In some embodiments, the survival is prolonged for at leastabout 2, 3, 4, 5, 6, 12, 24, or more months.

In some embodiments, there is provided a method of prolonging survival(such as disease free survival, progression-free survival, orcystectomy-free survival) in an individual having bladder cancer,comprising intravesically administering to the individual an oncolyticvirus (such as CG0070), wherein the oncolytic virus comprises a viralvector comprising a tumor cell-specific promoter operably linked to aviral gene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy). In some embodiments, the method further comprisesadministering to the individual a transduction enhancing agent (such asDDM) prior to the administration of the oncolytic virus. In someembodiments, the oncolytic virus is administered at a dose of about1×10⁸ to about 1×10¹⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, the oncolytic virus is administeredweekly. In some embodiments, the oncolytic virus is administered forabout 1 week to about 6 weeks (such as about 3 weeks or about 6 weeks).In some embodiments, the bladder cancer is CIS, CIS+Ta, CIS+T1, Ta, orT1 grade bladder cancer.

In some embodiments, the method improves quality of life in theindividual. In some embodiments, the individual does not need acystectomy (such as partial cystectomy or radical cystectomy) afterreceiving the viral therapy of the present application for at leastabout any one of 3, 6, 9, 12, 18, 24, 30, 36, 42, 48, 54, 60 or moremonths. In some embodiments, because the method of the presentapplication delays or obviates the need for radical cystectomy in theindividual, the individual enjoys improved quality of life compared toother bladder CIS patients because the individual does not suffer fromundesirable side effects due to reconstructive surgery after radicalcystectomy.

In some embodiments, there is provided a method of improving quality oflife in an individual having bladder CIS (such as pure CIS), comprisingintravesically administering to the individual an oncolytic virus (suchas CG0070), wherein the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy). In some embodiments, the method further comprisesadministering to the individual a transduction enhancing agent (such asDDM) prior to the administration of the oncolytic virus. In someembodiments, the oncolytic virus is administered at a dose of about1×10⁸ to about 1×10¹⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, the oncolytic virus is administeredweekly. In some embodiments, the oncolytic virus is administered forabout 1 week to about 6 weeks (such as about 3 weeks or about 6 weeks).In some embodiments, the bladder cancer is CIS, CIS+Ta, CIS+T1, Ta, orT1 grade bladder cancer.

In some embodiments, there is provided a method of treating bladdercancer in an individual without subjecting the individual to cystectomy(such as radical cystectomy), comprising intravesically administering tothe individual an oncolytic virus (such as CG0070), wherein theoncolytic virus comprises a viral vector comprising a tumorcell-specific promoter operably linked to a viral gene essential forreplication of the oncolytic virus, and a heterologous gene encoding animmune-related molecule. In some embodiments, the oncolytic viruscomprises a tumor-selective promoter. In some embodiments, the oncolyticvirus is not administered in conjunction with an immune checkpointmodulator. In some embodiments, the oncolytic virus is administered as asingle therapeutic agent. In some embodiments, the individual isunresponsive or has disease reoccurrence subsequent to BCG treatment. Insome embodiments, the individual has refused or is ineligible forcystectomy (such as radical cystectomy). In some embodiments, the methodfurther comprises administering to the individual a transductionenhancing agent (such as DDM) prior to the administration of theoncolytic virus. In some embodiments, the oncolytic virus isadministered at a dose of about 1×10⁸ to about 1×10¹⁴ viral particles(such as about 1×10¹² viral particles). In some embodiments, theoncolytic virus is administered weekly. In some embodiments, theoncolytic virus is administered for about 1 week to about 6 weeks (suchas about 3 weeks or about 6 weeks). In some embodiments, the bladdercancer is CIS, CIS+Ta, CIS+T1, Ta, or T1 grade bladder cancer.

In some embodiments, there is provided a bladder-preserving method oftreating bladder CIS (such as pure CIS) in an individual, comprisingintravesically administering to the individual an oncolytic virus (suchas CG0070), wherein the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus comprises a tumor-selectivepromoter. In some embodiments, the oncolytic virus is not administeredin conjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy). In some embodiments, the method further comprisesadministering to the individual a transduction enhancing agent (such asDDM) prior to the administration of the oncolytic virus. In someembodiments, the oncolytic virus is administered at a dose of about1×10⁸ to about 1×10¹⁴ viral particles (such as about 1×10¹² viralparticles). In some embodiments, the oncolytic virus is administeredweekly. In some embodiments, the oncolytic virus is administered forabout 1 week to about 6 weeks (such as about 3 weeks or about 6 weeks).In some embodiments, the bladder cancer is CIS, CIS+Ta, CIS+T1, Ta, orT1 grade bladder cancer.

Treatment Regimens

The intravesical administration of the oncolytic virus provide a uniqueopportunity of a relatively convenient yet effective intravesical tumorexposure to the oncolytic virus, as well as a potentially reducedtoxicity to other tissues. Suitable dosages for the oncolytic virusdepend on factors such as the nature of the oncolytic virus, type of thebladder carcinoma in situ being treated, and routes of administration.As used herein, “particles” as related to an oncolytic virus mean thecollective number of physical singular units of the oncolytic virus.This number can be converted to, or is equivalent to, another numbermeaning infectious titer units, e.g., plaque forming unit (pfu) orinternational unit, by infectivity assays as known in the art. In someembodiments, the oncolytic virus is administered at a dose of about anyone of 1×10⁵ particles, 1×10⁶ particles, 1×10⁷ particles, 1×10⁸particles, 1×10⁹ particles, 1×10¹⁰ particles, 2×10¹⁰ particles, 5×10¹⁰particles, 1×10¹¹ particles, 2×10¹¹ particles, 5×10¹¹ particles, 1×10¹²particles, 2×10¹² particles, 5×10¹² particles, 1×10¹³ particles, 2×10¹³particles, 5×10¹³ particles, 1×10¹⁴ particles, or 1×10¹¹ particles. Insome embodiments, the oncolytic virus is administered at a dose of anyone of about 1×10⁵ particles to about 1×10⁶ particles, about 1×10⁶particles to about 1×10⁷ particles, about 1×10⁷ particles to about 1×10⁸particles, about 1×10⁸ particles to about 1×10⁹ particles, about 1×10⁹particles to about 1×10¹⁰ particles, about 1×10¹⁰ particles to about1×10¹¹ particles, about 1×10¹¹ particles to about 5×10¹¹ particles,about 5×10¹¹ particles to about 1×10¹² particles, about 1×10¹² particlesto about 2×10¹² particles, about 2×10¹² particles to about 5×10¹²particles, about 5×10¹² particles to about 1×10¹³ particles, about1×10¹³ particles to about 1×10¹⁴ particles, or about 1×10¹⁴ particles toabout 1×10¹⁵ particles.

In some embodiments, the oncolytic virus is administered daily. In someembodiments, the oncolytic virus is administered is administered atleast about any one of 1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e., daily) aweek. In some embodiments, the oncolytic virus is administered weekly.In some embodiments, the oncolytic virus is administered weekly withoutbreak; weekly, two out of three weeks; weekly three out of four weeks;once every two weeks; once every 3 weeks; once every 4 weeks; once every6 weeks; once every 8 weeks, monthly, or every two to 12 months. In someembodiments, the intervals between each administration are less thanabout any one of 6 months, 3 months, 1 month, 20 days, 15, days, 12days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2days, or 1 day. In some embodiments, the intervals between eachadministration are more than about any one of 1 month, 2 months, 3months, 4 months, 5 months, 6 months, 8 months, or 12 months. In someembodiments, there is no break in the dosing schedule. In someembodiments, the interval between each administration is no more thanabout a week.

The administration of the oncolytic virus can be over an extended periodof time, such as from about a month up to about seven years. In someembodiments, the oncolytic virus is administered over a period of atleast about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24,30, 36, 48, 60, 72, or 84 months. In some embodiments, the oncolyticvirus is administered over a period of at least 4 weeks or 6 weeks. Insome embodiments, the oncolytic virus is administered weekly for fourweeks every 3 months. In some embodiments, the oncolytic virus isadministered weekly for 6 weeks every 3 months.

In some embodiments, each treatment course comprises weeklyadministration of the oncolytic virus for about 3 or about 6 weeks. Insome embodiments, the interval between each course of treatment is atleast about any one of 1, 2, 3, 4, 5, 6, or more months. In someembodiments, the interval between each course of treatment is no morethan about any one of 6, 5, 4, 3, 2, 1 months or less. In someembodiments, the treatment course is repeated at least once, such as atleast about any of 1, 2, 3, 4, 5, 6, or more times. In some embodiments,the individual is treated with an initial course of the oncolytic virus,followed by a maintenance course. In some embodiments, the initialcourse and the maintenance course have the same dose and schedule. Insome embodiments, the initial course and the maintenance course havedifferent doses and/or schedules.

In some embodiments, the oncolytic virus is administered by instillationas a solution via a catheter. In some embodiments, the total volume ofthe solution used for the intravesical installation is about any of 1mL, 10 mL, 50 mL, 75 mL, 100 mL, 125 mL, 150 mL, 200 mL, 250 mL, 300 mL,400 mL or 500 mL. In some embodiments, the total volume of the solutionused for the intravesical installation is any of about 1 mL to about 10mL, about 10 mL to about 50 mL, about 50 mL to about 75 mL, about 75 mLto about 100 mL, about 100 mL to about 125 mL, about 75 mL to about 125mL, about 100 mL to about 150 mL, about 150 mL to about 200 mL, about200 mL to about 300 mL, about 300 mL to about 400 mL, about 400 mL toabout 500 mL, about 50 mL to about 500 mL, about 50 mL to about 250 mL,or about 100 mL to about 250 mL.

In some embodiments, the oncolytic virus is administered at a dose ofabout 1×10⁸ to about 1×10¹⁵ particles (such as about 1×10¹¹ to about1×10¹⁴ particles, for example about 1×10¹² particles). In someembodiments, the oncolytic virus is administered at a volume of about 50to about 500 mL (such as about 100 mL) by instillation. In someembodiments, the oncolytic virus is administered at a dose of about1×10¹² in about 50 mL.

The solution of the oncolytic virus may be retained in the bladder for acertain amount of time before voiding, in order to achieve uniformdistribution or sufficient exposure of the oncolytic virus among thebladder tumor cells. In some embodiments, the solution is retained inthe bladder of the individual for at least about any of 5 minutes, 10minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, or more. In some embodiments, the solution is retained in thebladder of the individual for any of about 5 minutes to about 10minutes, about 10 minutes to about 15 minutes, about 10 minutes to about20 minutes, about 20 minutes to about 30 minutes, about 30 minutes toabout 45 minutes, about 45 minutes to about 50 minutes, about 50 minutesto about 1 hour, about 5 minutes to about 15 minutes, about 10 minutesto about 30 minutes, about 30 minutes to about 1 hour, or about 1 hourto about 2 hours. In some embodiments, the oncolytic virus (e.g.,CG0070) is retained in the bladder of the individual for about 45minutes to about 50 minutes. In some embodiments, the efficiency of theintravesical administration of the oncolytic virus is further enhancedby a pretreatment comprising intravesical administration of an effectiveamount of a transduction enhancing agent, such as DDM.

The methods described herein may further comprise a step ofintravesically administering to the individual a pretreatmentcomposition prior to the administration of the oncolytic virus. In someembodiments, the pretreatment composition comprises a transductionenhancing agent, such as N-Dodecyl-β-D-maltoside (DDM). DDM is anonionic surfactant comprised of a maltose derivatized with a singletwelve-carbon chain, and acts as a mild detergent and solubilzing agent.It has been used as a food additive and is known to enhance mucosalsurface permeation in rodents, probably due to its effect on membraneassociated GAG and tight junctions.

In some embodiments, there is provided a method of treating bladdercancer in an individual, comprising: (1) intravesically administering tothe individual a transduction enhancing agent (such as DDM); andsubsequently (2) intravesically administering to the individual anoncolytic virus (such as CG0070), wherein the oncolytic virus comprisesa viral vector comprising a tumor cell-specific promoter operably linkedto a viral gene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the oncolytic virus comprises a tumor-selective promoter.In some embodiments, the oncolytic virus is not administered inconjunction with an immune checkpoint modulator. In some embodiments,the oncolytic virus is administered as a single therapeutic agent. Insome embodiments, the individual is unresponsive or has diseasereoccurrence subsequent to BCG treatment. In some embodiments, theindividual has refused or is ineligible for cystectomy (such as radicalcystectomy). In some embodiments, the oncolytic virus is administered ata dose of about 1×10⁸ to about 1×10¹⁴ viral particles (such as about1×10¹² viral particles). In some embodiments, the oncolytic virus isadministered weekly. In some embodiments, the oncolytic virus isadministered for about 1 week to about 6 weeks (such as about 3 or about6 weeks). In some embodiments, the bladder cancer is CIS, CIS+Ta,CIS+T1, Ta, or T1 grade bladder cancer. In some embodiments, theindividual does not receive a resection prior to administration ofoncolytic virus. In some embodiments, the individual has Ta or T1bladder cancer and has not received a transurothelial resection ofbladder tumor (TURBT). In some embodiments, the bladder cancer is Ta orT1 non-resectable bladder cancer. In some embodiments, a saline wash isperformed prior to intravesically administering to the individual atransduction enhancing agent.

In some embodiments, provided herein is a method of treating bladdercancer (such as CIS bladder cancer or Ta or T1 grade bladder cancerwithout TURBT) comprising: (1) administering an intravesicular salinewash (2) administering an intravesicular wash with a transductionenhancing agent (such as DDM); (3) administering to the individual anintravesicular instillation of a transduction enhancing agent (such asDDM); and subsequently (4) intravesically administering to theindividual an oncolytic virus (such as CG0070), wherein the oncolyticvirus comprises a viral vector comprising a tumor cell-specific promoteroperably linked to a viral gene essential for replication of theoncolytic virus, and a heterologous gene encoding an immune-relatedmolecule. In some embodiments, the oncolytic virus comprises atumor-selective promoter. In some embodiments, the oncolytic virus isnot administered in conjunction with an immune checkpoint modulator. Insome embodiments, the oncolytic virus is administered as a singletherapeutic agent. In some embodiments, the individual is unresponsiveor has disease reoccurrence subsequent to BCG treatment. In someembodiments, the individual has refused or is ineligible for cystectomy(such as radical cystectomy). In some embodiments, the oncolytic virusis administered at a dose of about 1×10⁸ to about 1×10¹⁴ viral particles(such as about 1×10¹² viral particles). In some embodiments, theoncolytic virus is administered weekly. In some embodiments, theoncolytic virus is administered for about 1 week to about 6 weeks (suchas about 3 or about 6 weeks). In some embodiments, the bladder cancer isCIS, CIS+Ta, CIS+T1, Ta, or T1 grade bladder cancer. In someembodiments, the individual has Ta or T1 bladder cancer and has notreceived a transurothelial resection of bladder tumor (TURBT). In someembodiments, the bladder cancer is Ta or T1 non-resectable bladdercancer. In some embodiments, the individual does not receive a resectionprior to administration of oncolytic virus. In some embodiments thetransduction enhancing agent is retained in the bladder for 12 to 15minutes during the intravesicular instillation. In some embodiments, asaline rinse is administered following intravesicular instillation of atransduction enhancing agent and administering an oncolytic virus.

The pretreatment composition is administered intravesically. In someembodiments, the pretreatment composition comprises a solution of thetransduction enhancing agent (such as DDM). Suitable concentration ofthe pretreatment composition (such as DDM solution) include, but are notlimited to, about any one of 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,1%, 2%, 3%, 4%, or 5% of the transducing enchanting agent (such as DDM).In some embodiments, the pretreatment composition comprises any of about0.01% to about 0.05%, about 0.05% to about 0.1%, about 0.1% to about0.5%, about 0.5% to about 1%, about 1% to about 2%, about 2% to about3%, about 3% to about 4%, about 4% to about 5%, about 0.01% to about 1%,about 0.05% to about 2%, about 1% to about 5%, or about 0.1% to about 5%of the transduction enhancing agent (such as DDM).

Suitable dosages for the pretreatment composition (such as DDM) include,but are not limited to, about any of 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5mg/kg, 2 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 100mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg,0.1 mg/kg to 0.5 mg/kg, 0.5 mg/kg to 1 mg/kg, 1 mg/kg to 2 mg/kg, 2mg/kg to 5 mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kg to 25 mg/kg, 25 mg/kg to50 mg/kg, 50 mg/kg to 100 mg/kg, 100 mg/kg to 150 mg/kg, 150 mg/kg to200 mg/kg, 200 mg/kg to 250 mg/kg, 250 mg/kg to 500 mg/kg, or 0.5 mg/kgto about 5 mg/kg. In some embodiments, a suitable dosage for thepretreatment composition is about any one of 0.1 g, 0.2 g, 0.5 g, 0.75g, 1 g, 1.5 g, 2 g, 2.5 g, 5 g, or 10 g of the transduction enhancingagent (such as DDM).

In some embodiments, the pretreatment step is carried out by contactingthe luminal surface of the bladder in the individual with thepretreatment composition prior to the administration of the oncolyticvirus. For example, the pretreatment composition may comprise about0.01% to about 0.5% (such as 0.05 to about 0.2%, for example about 0.1%)of the transduction enhancing agent (such as DDM). In some embodiments,the total volume of the pretreatment composition (such as DDM) is about10 mL to about 1000 mL (such as about 10 mL to about 100 mL, about 100mL to about 500 mL, or about 500 mL to about 1000 mL). In someembodiments, the pretreatment composition comprises about 0.1% DDM. Insome embodiments, a suitable dosage for the pretreatment composition isabout any one of 0.1 g, 0.2 g, 0.5 g, 0.75 g, 1 g, 1.5 g, 2 g, 2.5 g, 5g, or 10 g of the transduction enhancing agent (such as DDM). In someembodiments, the effective amount of the pretreatment composition isabout 1 g of DDM (e.g., 100 mL of 0.1% DDM solution).

In some embodiments, the pretreatment composition (such as DDM) isadministered immediately (such as no more than 5 minutes) prior to theadministration of the oncolytic virus.

In some embodiments, the pretreatment composition (such as DDM) isadministered no more than about any of 5 minutes, 10 minutes, 15minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 90 minutes, 2hours, 3 hours or 4 hours before the administration of the oncolyticvirus. In some embodiments, the pretreatment composition (such as DDM)is administered no more than about 2 hours before the administration ofthe oncolytic virus. In some embodiments, the pretreatment composition(such as DDM solution) is retained in the bladder for at least about anyone of 5 minutes, 10 minutes, 15 minutes, or 20 minutes. In someembodiments, the pretreatment composition (such as DDM solution) isretained in the bladder for any of about 5 minutes to about 10 minutes,about 10 minutes to about 15 minutes, about 12 minutes to about 15minutes, about 15 minutes to about 20 minutes, or about 10 minutes toabout 20 minutes. In some embodiments, the pretreatment composition(such as DDM solution) is retained in the bladder for about 12 minutesto about 15 minutes.

In some embodiments, the pretreatment step is carried out by contactingthe luminal surface of the bladder in the individual with thepretreatment composition prior to the administration of the oncolyticvirus. In some embodiments, the method further comprises washing theluminal surface of the bladder contact with the pretreatmentcomposition. In some embodiments, the method further comprises washingthe luminal surface of the bladder after contacting the bladder with thepretreatment composition prior to the administration of the oncolyticvirus.

In some embodiments, the pretreatment step comprises intravesicallyinstilling 75 mL or 100 mL of a pretreatment composition (such as a DDMsolution).

In some embodiments, the individual is a human individual. In someembodiments, the individual being treated for bladder carcinoma in situhas been identified as having one or more of the conditions describedherein. Identification of the conditions as described herein by askilled physician is routine in the art (e.g., via blood tests, X-rays,ultrasound, CT scans, PET scans, PET/CT scans, MRI scans, PET/MRI scans,nuclear medicine radioisotope scans, endoscopy, biopsy, angiography,CT-angiography, etc.) and may also be suspected by the individual orothers, for example, due to tumor growth, hemorrhage, ulceration, pain,enlarged lymph nodes, cough, jaundice, swelling, weight loss, cachexia,sweating, anemia, paraneoplastic phenomena, thrombosis, etc. In someembodiments, the individual is selected for any one of the treatmentmethods described herein based on any one or more of a number of riskfactors and/or diagnostic approaches appreciated by the skilled artisan,including, but not limited to, genetic profiling, family history,medical history (e.g., appearance of related conditions and viralinfection history), lifestyle or habits.

Oncolytic Virus

The methods and compositions described herein are related to oncolyticviruses, for example, oncolytic adenovirus. The oncolytic virus may be anaturally occurring virus, or a genetically modified virus, for examplean attenuated virus, and/or a virus with additional favorable features(e.g., preferential replication in cancer cells, or encoding animmune-related molecule).

Exemplary viruses that are suitable for use in the present inventioninclude, but are not limited to, adenovirus, for example, H101(ONCOCRINE®), CG-TG-102 (Ad5/3-D24-GM-CSF), and CG0070; herpes simplexvirus, for example, Talimogene laherparapvec (T-VEC) and HSV-1716(SEPREHVIR®); reo virus, for example, REOLYSIN®; vaccinia virus, forexample, JX-594; Seneca valley virus, for example, NTX-010 and SVV-001;newcastle disease virus, for example, NDV-NSI and GL-ONC1; polio virus,for example, PVS-RIPO; measles virus, for example, MV-NIS; coxsackievirus, for example, CAVATAK™; vesicular stomatitis virus; maraba andrhabdoviruses; parvovirus and mumps virus.

In some embodiments, the oncolytic virus is a wild type oncolytic virus.In some embodiments, the oncolytic virus is genetically modified. Insome embodiments, the oncolytic virus is attenuated (for example throughmultiple passages, inactivation or genetic modification). In someembodiments, the oncolytic virus is replication competent. In someembodiments, the oncolytic virus preferentially replicates in a cancercell, such as a cancer cell defective in the Rb pathway.

In some embodiments, the oncolytic virus (such as oncolytic adenovirus)comprises a viral vector comprising a tumor cell-specific promoteroperably linked to a viral gene essential for replication of theoncolytic virus. In some embodiments, the tumor-specific promoter is anE2F-1 promoter, such as a human E2F-1 promoter or an E2F-1 promotercomprising the nucleotide sequence set forth in SEQ ID NO:1 as shownbelow. In some embodiments, the viral gene essential for replication ofthe oncolytic virus is selected from the group consisting of E1A, E1B,and E4.

In some embodiments, the oncolytic virus (such as oncolytic adenovirus)comprises a viral vector comprising a tumor-selective promoter operablylinked to a viral gene essential for replication of the oncolytic virus.In some embodiments, the tumor-selective promoter is an E2F-1 promoter,such as a human E2F-1 promoter or an E2F-1 promoter comprising thenucleotide sequence set forth in SEQ ID NO:1 as shown below. In someembodiments, the viral gene essential for replication of the oncolyticvirus is selected from the group consisting of E1A, E1B, and E4.

(E2F-1 promoter) SEQ ID NO: 1gggcccaaaattagcaagtgaccacgtggttctgaagccagtggcctaaggaccacccttgcagaaccgtggtctccttgtcacagtctaggcagcctctggcttagcctctgtttctttcataacctttctcagcgcctgctctgggccagaccagtgttgggaggagtcgctactgagctcctagattggcaggggaggcagatggagaaaaggagtgtgtgtggtcagcattggagcagaggcagcagtgggcaatagaggaagtgagtaaatccttgggagggctccctagaagtgatgtgttttctttttttgttttagagacaggatctcgctctgtcgcccaggctggtgtgcagtggcatgatcatagctcactgcagcctcgacttctcgggctcaagcaatcctcccacctcagcctcccaagtagctgggactacgggcacacgccaccatgcctggctaatttttgtattttttgtagagatgggtcttcaccatgttgatcaggctggtctcgaactcctgggctcatgcgatccaccccgccagctgattacagggattccggtggtgagccaccgcgcccagacgccacttcatcgtattgtaaacgtctgttacctttctgttcccctgtctactggactgtgagctccttagggccacgaattgaggatggggcacagagcaagctctccaaacgtttgttgaatgagtgagggaatgaatgagttcaagcagatgctatacgttggctgttggagattttggctaaaatgggacttgcaggaaagcccgacgtccccctcgccatttccaggcaccgctcttcagcttgggctctgggtgagcgggatagggctgggtgcaggattaggataatgtcatgggtgaggcaagttgaggatggaagaggtggctgatggctgggctgtggaactgatgatcctgaaaagaagaggggacagtctctggaaatctaagctgaggctgttgggggctacaggttgagggtcacgtgcagaagagaggctctgttctgaacctgcactatagaaaggtcagtgggatgcgggagcgtcggggcggggcggggcctatgttcccgtgtccccacgcctccagcaggggacgcccgggctgggggcggggagtcagaccgcgcctggtaccatccggacaaagcctgcgcgcgccccgccccgccattggccgtaccgccccgcgccgccgccccatcccgcccctcgccgccgggtccggcgcgttaaagccaataggaaccgccgccgttgttcccgtcacggacggggcagccaattgtggcggcgctcggcggctcgtggctctttcgcggcaaaaaggatttggcgcgtaaaagtggccgggactttgcaggcagcggcggccgggggcggagcgggatcgagccctcgccgaggcctgccgccatgggcccgcgccgccgccgccgcctgtcacccgggccgc gcgggccgtgagcgtcatg

In some embodiments, the oncolytic virus (such as oncolytic adenovirus)comprises a viral vector comprising a tumor cell-specific promoteroperably linked to a viral gene essential for replication of theoncolytic virus and a nucleic acid encoding an immune-related molecule(such as cytokine or chemokine) operably linked to a viral promoter. Insome embodiments, the tumor-specific promoter is an E2F-1 promoter, suchas a human E2F-1 promoter or an E2F-1 promoter comprising the nucleotidesequence set forth in SEQ ID NO:1. In some embodiments, the viral geneessential for replication of the oncolytic virus is selected from thegroup consisting of E1A, E1B, and E4. In some embodiments, the viralpromoter operably linked to the nucleic acid encoding the immune-relatedmolecule is the E3 promoter. In some embodiments, the immune-relatedmolecule is GM-CSF.

In some embodiments, the oncolytic virus (such as oncolytic adenovirus)comprises a viral vector comprising a tumor cell-selective promoteroperably linked to a viral gene essential for replication of theoncolytic virus and a nucleic acid encoding an immune-related molecule(such as cytokine or chemokine) operably linked to a viral promoter. Insome embodiments, the tumor-selective promoter is an E2F-1 promoter,such as a human E2F-1 promoter or an E2F-1 promoter comprising thenucleotide sequence set forth in SEQ ID NO:1. In some embodiments, theviral gene essential for replication of the oncolytic virus is selectedfrom the group consisting of E1A, E1B, and E4. In some embodiments, theviral promoter operably linked to the nucleic acid encoding theimmune-related molecule is the E3 promoter. In some embodiments, theimmune-related molecule is GM-CSF.

In some embodiments, the oncolytic virus is an adenovirus serotype 5,wherein the endogenous Ela promoter and E3 19kD coding region of anative adenovirus is replaced by the human E2F-l promoter and a nucleicacid encoding an immune-related molecule (such as cytokine or chemokine,for example, GM-CSF). In some embodiments, the tumor-specific promoteris a human E2F-1 promoter or an E2F-1 promoter comprising the nucleotidesequence set forth in SEQ ID NO:1.

In some embodiments, the oncolytic virus is CG0070, an adenovirusserotype 5 which has an E2F promoter at the Ela gene and a GM-CSFexpression at the E3 gene.

CG0070 is a conditionally replicating oncolytic adenovirus (serotype 5)designed to preferentially replicate in and kill Rb pathway-defectivecancer cells. This vector is transcriptionally regulated by a promoter(e.g., E2F-1 promoter) that is up-regulated in Rb-pathway-detectivetumor cells. In approximately 85% of all cancers, one or more genes ofthe Rb pathway, such as the tumor suppressor Rb gene, are mutated. Inaddition to its restricted propagation, CG0070 also encodes the humancytokine GM-CSF, which is expressed selectively in the infected tumorcells to stimulate immune responses against uninfected distant (such asmetastases) and local tumor foci.

The genomic structure of the oncolytic adenoviral vector CG0070 is shownschematically in FIG. 1 . Products of the adenoviral early E1A gene areessential for efficient expression of other regions of the adenoviralgenome. CG0070 has been engineered to express the E1A gene under controlof the human E2F-1 promoter, which provides tumor specificity to the E1Agene product. To protect from transcriptional read-through activatingE1A expression, a polyadenylation signal (PA) was inserted 5′ of theE2F-1 promoter. CG0070 includes the entire wild type E3 region exceptfor the 19kD-coding region. A direct comparison of E3-containing toE3-deleted oncolytic adenovirus vectors showed superiority ofE3-containing vectors in tumor spread and efficacy. In place of the 19kDgene, CG0070 carries the cDNA for human GM-CSF under the control of theendogenous E3 promoter (E3P). Since the E3 promoter is in turn activatedby E1A, both viral replication and GM-CSF expression are ultimatelyunder the control of the E2F-1 promoter. The rest of the viral vectorbackbone, including the E2, E4, late protein regions and invertedterminal repeats (ITRs), is identical to the wild type Ad5 genome.

CG0070 is manufactured in HeLa-S3 cells, and released from infectedHeLa-S3 cells by detergent lysis. CG0070 is purified from the lysate bychromatography, and then formulated in 5% sucrose, 10 mM Tris, 0.05%polysorbate-80, 1% glycine, 1 mM magnesium chloride, pH 7.8.

CG0070 is supplied as a sterile, slightly opalescent, frozen liquid instoppered glass vials. The particle concentration per mL (vp/mL) isstated on the Certificate of Analysis for each lot of CG0070.

CG0070 has additional potential anti-tumor activity in that it carriesthe cDNA for human GM-CSF, a key cytokine for generating long-lastinganti-tumor immunity. Thus, CG0070 is a selectively replicating oncolyticvector with the potential for attacking the tumor by two mechanisms:direct cytotoxicity as a replicating vector and induction of a hostimmune response. In vitro and in vivo studies have been conducted tocharacterize the tumor selectivity and anti-tumor activity and safety ofCG0070. See, for example, U.S. patent application publication No.US20150190505, which incorporated herein by reference in its entirety.

III Pharmaceutical Compositions, Kits, and Articles of Manufacture

In another aspect, there are provided kits, unit dosages, and articlesof manufacture useful for any one of the methods described herein.

In some embodiments, there is provided a pharmaceutical compositioncomprising an oncolytic virus (such as CG0070) and a pharmaceuticallyacceptable carrier, wherein the oncolytic virus comprises a viral vectorcomprising a tumor cell-specific promoter operably linked to a viralgene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. Thepharmaceutical composition may be used for treating bladder CIS (such aspure CIS) according to any one of the methods described herein.

Suitable pharmaceutical carriers include sterile water; saline,dextrose; dextrose in water or saline; condensation products of castoroil and ethylene oxide combining about 30 to about 35 moles of ethyleneoxide per mole of castor oil; liquid acid; lower alkanols; oils such ascorn oil; peanut oil, sesame oil and the like, with emulsifiers such asmono- or di-glyceride of a fatty acid, or a phosphatide, e.g., lecithin,and the like; glycols; polyalkylene glycols; aqueous media in thepresence of a suspending agent, for example, sodiumcarboxymethylcellulose; sodium alginate; poly(vinylpyrolidone); and thelike, alone, or with suitable dispensing agents such as lecithin;polyoxyethylene stearate; and the like. The carrier may also containadjuvants such as preserving stabilizing, wetting, emulsifying agentsand the like together with the penetration enhancer. The final form maybe sterile and may also be able to pass readily through an injectiondevice such as a hollow needle. The proper viscosity may be achieved andmaintained by the proper choice of solvents or excipients. Thepharmaceutical carrier may include active or passive excipients for drugdelivery, such as polymer and non-polymer systems. Otherpharmaceutically acceptable carriers and their formulation are describedin standard formulation treatises, e.g., Remington's PharmaceuticalSciences by E. W. Martin.

The pharmaceutical compositions described herein may include otheragents, excipients, or stabilizers to improve properties of thecomposition. Examples of suitable excipients and diluents include, butare not limited to, lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water, saline solution, syrup, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate and mineral oil. Insome embodiments, the pharmaceutical composition is formulated to have apH in the range of about 4.5 to about 9.0, including for example pHranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, orabout 6.5 to about 7.0. In some embodiments, the pharmaceuticalcomposition can also be made to be isotonic with blood by the additionof a suitable tonicity modifier, such as glycerol.

In some embodiments, there is provided a kit for treating bladdercarcinoma in situ (such as pure CIS) in an individual, comprising anoncolytic virus (such as CG0070), wherein the oncolytic virus comprisesa viral vector comprising a tumor cell-specific promoter operably linkedto a viral gene essential for replication of the oncolytic virus, and aheterologous gene encoding an immune-related molecule. In someembodiments, the kit further comprises a pretreatment compositioncomprising a transduction enhancing agent, such asN-Dodecyl-β-D-maltoside (DDM). In some embodiments, the kit furthercomprises devices, materials, and/or instructions for carrying out anyone of the methods described above. Medical device for intravesicaldelivery may include a catheter, for example, a RUSCH® 173430 FoleyCatheter & BARD® LUBRI-SIL® Foley Catheter #70516SI.

The instructions relating to the use of the oncolytic virus (such asCG0070) generally include information as to dosage, dosing schedule, androute of administration for the intended treatment. The containers maybe unit doses, bulk packages (e.g., multi-dose packages) or sub-unitdoses. For example, kits may be provided that contain sufficient dosagesof the oncolytic virus as disclosed herein to provide effectivetreatment of an individual for an extended period, such as any of aweek, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5months, 7 months, 8 months, 9 months, 12 months or more. Kits may alsoinclude multiple unit doses of the oncolytic virus and instructions foruse, packaged in quantities sufficient for storage and use inpharmacies, for example, hospital pharmacies and compounding pharmacies.

The kits of the invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed MYLAR® or plastic bags), and the like. Kits mayoptionally provide additional components such as buffers andinterpretative information. The present application thus also providesarticles of manufacture, which include vials (such as sealed vials),bottles, jars, flexible packaging, and the like.

The article of manufacture can comprise a container and a label orpackage insert on or associated with the container. Suitable containersinclude, for example, bottles, vials, syringes, etc. The containers maybe formed from a variety of materials such as glass or plastic.Generally, the container holds a composition which is effective fortreating bladder CIS as described herein, and may have a sterile accessport. The label or package insert indicates that the composition is usedfor treating the particular condition in an individual. The label orpackage insert will further comprise instructions for administering thecomposition to the individual. Articles of manufacture and kitscomprising combination therapies described herein are also contemplated.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In someembodiments, the package insert indicates that the composition is usedfor treating bladder carcinoma in situ (such as pure CIS).

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

EXAMPLES

The examples below are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway. The following examples and detailed description are offered by wayof illustration and not by way of limitation.

Example 1: A Phase II Clinical Trial of CG0070 for TreatingBCG-Unresponsive Non-Muscle-Invasive Bladder Cancer (NMIBC)

This example describes an open-label, single-arm, Phase II, multicenterstudy of the safety and efficacy of intravesical CG0070 monotherapy inpatients with non-muscle invasive bladder carcinoma who have failed BGtherapy and refused cystectomy. Most patients with NMIBC (Cis, Cis withTa and/or T1, high grade Ta or T1 with frequent or uncontrolledrecurrences) who have failed BCG intravesical therapy (standard of care)usually have no other choice but to proceed to cystectomy. Cystectomy isa surgery associated with major morbidity, mortality and quality of lifeissues. Morbidity and long term tedious medical care are associated withthe rest of the patient's life span after cystectomy. Most patients atthe NMIBC stages do not show signs of disease progression into themuscle layer or of metastasis, making surgery a very difficult decision.There is unmet need for therapeutic alternatives among this patientpopulation.

CG0070 is a replication selective oncolytic adenovirus that destroysbladder tumor cells through their defective retinoblastoma (Rb) pathway.Prior reports of intravesical CG0070 have shown promising activity inpatients with high-grade NMIBC who previously did not respond to BCG.However, limited accrual has hindered analysis of efficacy, particularlyfor pathologic subsets. Interim analysis at 6 months from the firstintravesical administration of CG0070 was conducted to investigate theefficacy of CG0070 monotherapy in patients having bladder carcinoma insitu (CIS) with or without concurrent papillary carcinoma at Ta or T1stage.

Experimental Design

In the study, each patient received intravesical CG0070 at a dose of1×10² viral particles weekly for six weeks. Patients achieved a partialresponse or a complete response at 6 months post first intravesicalintervention were maintained with the same treatment courses of sixweekly intravesical instillation of CG0070. Patients were followed every3 months for 24 months. At the 6-month follow up, patients weresubjected to cystoscopy, urine cytology and biopsy. If no suspiciouslesions were found, random biopsy of the bladder was obtained from thepatients. Biopsy samples were reviewed by local and centralpathologists, and treatment response was determined using both biopsyreadings.

The primary outcome of the study measures durable complete responseproportion (DCR) at the 18-month time point from the date of the firstintravesical administration of CG0070. DCR is defined as the proportionof patients who experience a durable complete response lasting at least6 months or longer (first interim analysis), at least 12 months orlonger from the initial confirmed complete response date, and at least18 months from the date of the first intravesical intervention.Secondary outcome measures include: cystectomy free survival at 18months after the first intravesical treatment, complete responsesurvival at 18 months after the first intravesical treatment,progression free survival at 18 months after the first intravesicaltreatment, time to progression to muscle invasive disease at 18 monthsafter the first intravesical treatment, overall survival at 18 monthsand 24 months after the first intravesical treatment, PD-L1 statuschanges between pre-intervention and post intervention at eithercystectomy or at biopsy up to 24 months, PD-L1 status of cancer cellsand immune cells at tumor site by IHC, organ confined diseaseproportions at cystectomy, patient proportions with no cancer cells inregional lymph nodes at cystectomy, complete response proportions at 24months after the first intravesical treatment, proportions of patientswith a complete response of at least 12-month duration, diseaseregression proportions at 24 months after the first intravesicaltreatment, and proportions of patients with a partial response and/or acomplete response of less than 12-month duration.

Patients must meet all of the following conditions to be eligible forthe study:

1. 18 years of age or older, including adults and seniors;

2. Patients must have pathologically confirmed non-muscle invasivebladder cancer (NMIBC) high grade disease (HG), as defined by the 2004WHO classification system;

3. Patients must have no evidence of muscle invasive disease;

4. Patients must be able to provide a sufficient biopsy sample to thecentral pathologist for histopathologically confirmed, transitional cell(urothelial) carcinoma. Urothelial tumors with mixed histology (but with<50% variant) are eligible;

5. Patients must have received at least two or more prior courses ofintravesical therapy per recommended schedules. BCG must have been oneof the prior therapies administered;

6. Patients can have either failed BCG induction therapy within asix-month period or have been successfully treated with BCG, butsubsequently found to have recurrence. The first standard course ofintravesical BCG therapy must include at least six weekly treatments(allowable range of instillations per course is 4-9). The second courseof BCG therapy must include at least two weekly treatments;7. Patients have either Cis or Cis with Ta and/or T1 disease atenrollment or in the past. For those patients with only Ta or T1 diseaseat enrollment AND with no history of Cis, they must have diseaserecurrence either must occur within 12 months of the most recentintravesical therapy of any kind, or disease recurrence within 18 monthsof BCG maintenance or disease recurrence within 24 months of BCGinduction;8. T1 patients need to have evidence of muscle included in their latestbiopsy; and if not a re-TURBT has to be done prior to enrollment;9. Radical cystectomy has been declined by the patient in a signedspecial section of the informed consent, whereby there is a clearexplanation by the investigator to the subject that a delay ofcystectomy may increase his/her chance of disease progression, theresults of which may lead to serious and life threatening consequences;10. Patients must be able to enter into the study within ten weeks oftheir most recent diagnostic procedure, which is usually a diagnosticbiopsy, a transurethral resection of bladder tumor (TURBT) procedure orpositive urine cytology;11. Eastern Cooperative Oncology Group (ECOG) performance status <2;12. Not pregnant or lactating;13. Patients with child bearing potential must agree to use adequatecontraception;14. Agree to study specific informed consent and HIPAA authorization forrelease of personal health information; and15. Adequate baseline CBC, renal and hepatic function. Parametersdescribed as WBC>3000 cells/mm³, ANC>1,000 cells/mm³, hemoglobin>9.5g/dL, and platelet count >100,000 cells/mm³; Adequate renal function:serum creatinine <2.5 mg/dL; Bilirubin, AST and ALT not more than 2×Upper Limits of Normal; PT/INR, PTT, and fibrinogen within institutionalacceptable limits; Absolute lymphocyte count >800/L before the firstdose of CG0070.

Patients who meet any of the following exclusion criteria are excludedfrom the study:

1. Previous systemic chemotherapy or radiation for bladder cancer. Note:Prior immunotherapy or intravesical (administered within the bladder)chemotherapy for superficial disease is acceptable;

2. History of anaphylactic reaction following exposure to humanized orhuman therapeutic monoclonal antibodies, hypersensitivity to GM-CSF oryeast derived products, clinically meaningful allergic reactions or anyknown hypersensitivity or prior reaction to any of the formulationexcipients in the study drugs;3. Known infection with HIV, HBV or HCV;4. Anticipated use of chemotherapy or radiotherapy not specified in thestudy protocol while on study;5. Any underlying medical condition that, in the Investigator's opinion,will make the administration of study vector hazardous to the patient,would obscure the interpretation of adverse events, or not permitadequate surgical resection;6. Systemic treatment on any investigational clinical trial within 28days prior to registration;7. Concurrent treatment with immunosuppressive or immunomodulatoryagents, including any systemic steroid (exception: inhaled or topicallyapplied steroids, and acute and chronic standard dose NSAIDs, arepermitted). Use of a short course (i.e., ≤1 day) of a glucocorticoid isacceptable to prevent a reaction to the IV contrast used for CT scans;8. Immunosuppressive therapy, including: cyclosporine, antithymocyteglobulin, or tacrolimus within 3 months of study entry;9. History of prior experimental cancer vaccine treatment (e.g.,dendritic cell therapy, heat shock vaccine) within the last year;10. History of stage III or greater cancer, excluding urothelial cancer.Basal or squamous cell skin cancers must have been adequately treatedand the subject must be disease-free at the time of registration.Subjects with a history of stage I or II cancer, must have beenadequately treated and have been disease-free for ≥2 years at the timeof registration;11. Progressive or persistent viral or bacterial infection;12. All infections must be resolved and the patient must remain afebrilefor seven days without antibiotics prior to being placed on study;13. Urinary tract infection, including particularly bladder infection,must be resolved prior to being placed on study; and14. Unwilling or unable to comply with the protocol or cooperate fullywith the investigator and site personnel.Interim Analysis ResultsInterim Analysis 1

At the interim analysis with an October 2016 cutoff date, thirty-sixpatients with residual high grade Ta, T1, or carcinoma in situ(CIS)+/−Ta/T1 had 6-month follow-up in this phase II single armmulticenter trial (NCT02365818). Inclusion criteria mandated receipt ofat least 2 prior courses of intravesical therapy for CIS, with at least1 of them being a course of BCG. Patients had either failed BCGinduction therapy within 6 months or had been successfully treated withBCG with subsequent recurrence. Complete response (CR) at 6 months wasdefined as absence of disease on cytology, cystoscopy, and randombiopsies.

As shown in FIG. 2 and Table 1 below, among the 36 patients in thisinterim analysis, there were 18 CIS, 4 CIS+Ta, 3 CIS+T1, 8 Ta, and 3 T1.Overall 6 month CR was 44%. Notably, 6-month CR for patients with pureCIS was 72.2%, CIS+/−Ta/T1 52%, CIS+Ta/T1 0%, pure Ta/T1 27%. Innon-responders with CIS, there were 4 patients (22%) with persistent CISat 6 months, and 1 (5.6%) that progressed to CIS+T1. No patients withpure T1 or CIS+Ta/T1 had 6-month CR. In patients with both CIS+Ta/T1(n=7), 5 had persistent Ta/T1+/−CIS, while 2 had CIS on biopsy at 6months.

TABLE 1 6-month CR results among evaluable patients. Stage No. CRs % CISonly (n = 18) 13 72 CIS + Ta (n = 4) 0 0 CIS + T1 (n = 3) 0 0 CIS +Ta/T1 (n = 25) 13 52 Ta (n = 8) 3 37 T1 (n = 3) 0 0 Ta/T1 (n = 11) 3 27Overall CR Rate (n = 36) 16 44

All treatment related adverse events (AEs) at 6 months were Grade 1-3,most commonly urinary: dysuria (47%), bladder spasms (44%), hematuria(36%), and urgency (33%). Immunologic treatment related AEs includedfatigue (11%) and chills (5.6%). Grade 3 treatment related AEs includeddysuria (5.6%) and hypotension (2.7%). There were no Grade 4/5 treatmentrelated AEs.

Interim Analysis 2

6-month clinical response results at the interim analysis with an Apr.14, 2017 cutoff date are summarized in Tables 2-5.

Patients with no evidence of malignancy according to either pathology orurine cytology examination, or in most recent pathology examination weredisqualified for the study. To determine the baseline stage of eachpatient, patient samples were evaluated by a local pathologist and acentral pathologist. If the local pathologist and the centralpathologist did not agree, but both found malignancy, then the stagesdetermined by both local and central pathologists were combined. If thelocal pathologist and the central pathologist did not agree and only onefound malignancy, then the patient was considered to have malignancyusing the stage determined by the pathologist who found malignancy. Ifthe patient samples were only evaluated by either the local pathologistor the central pathologist, then the single available reading was used.If neither pathologist found malignancy, but urine cytology waspositive, then the stage of the patient was annotated as “unknown.” Ifneither pathologist found malignancy, but urine cytology was absent ornegative, then the stage of the patient was annotated as “negative.”Pathology reports with “papillary” were entered as Ta+T1. Pathologyreports with “High grade UCC” was interpreted as “Ta”, unless invasioninto lamina propria (T1) or lamina muscularis (T2) was identified. Ifurine cytology was the only method of evaluation, and the result wasatypical or suspicious, and there was no pathological report, then thestage was annotated as “negative.” If urine cytology was the only methodof evaluation and a second urine cytology result was negative, then thetime point was considered negative.

Clinical response of the patients was determined using the same stagingrules as for the baseline stage assessment described above. Patientswith complete response (CR) include those who were assessed as“negative” at 6 month following CG0070 treatment. Patients withprogressive disease (PD) include those who had prior time pointassessment as “negative” and current time point assessment as not“negative,” and those who had progression to T2 or higher stage.

TABLE 2 Complete Response (CR) Rate Based on Stage in Intent to Treat(ITT) Population (n = 67*) at 6 Months Stage No. CRs % CIS (n = 31) 1445.2 CIS + Ta/T1 (n = 16) 4 25.0 CIS-Containing (n = 47) 18 38.3 Ta/T1(n = 19) 3 15.8 Unknown (n = 1) 0 0 All (n = 67) 21 31.3 *22 of 67patients considered Not Evaluable (NE) at 6 months and assumed not tohave a CR

TABLE 3 Overall Response at 6 Months in ITT Population (n = 67) ResponseNo. Patients % CR 21 31.3 Non-CR (SD + Regression) 12 17.9 PD 12 17.9 NE22 32.8

TABLE 4 Complete Responses (CRs) Based on Stage in Patients Achieving 6Month Evaluation (n = 45) Stage No. CRs % CIS (n = 24) 14 58.3 CIS +Ta/T1 (n = 12) 4 33.3 CIS-Containing (n = 36) 18 50 Ta/T1 (n = 9) 3 33.3All (n = 45) 21 46.7

TABLE 5 Overall Response in Patients Achieving 6 Month Evaluation (n =45) Response No. Patients % CR 21 46.7 Non-CR (SD + Regression) 12 26.7PD 12 26.7

This phase II study demonstrates that intravesical CG0070 yielded anoverall 46.7% complete response rate at 6 months with an acceptablelevel of toxicity for patients with high-risk BCG-unresponsive NMIBC.There is a particularly strong response and limited progression inpatients with pure CIS. Ongoing follow-up for this study will bevaluable in the BCG-unresponsive NMIBC population.

Final Results

A total of 67 patients having CIS, CIS+Ta, CIS+T1, Ta, or T1 stagebladder cancer were treated with intravesical C00070. Patients wereevaluated at 6 and 12 months for complete response, as described above.Table 7 shows the response rate to CG0070 for patients with BCGunresponsive, BCG refractory, and BCG relapsing bladder cancer. BCGunresponsive patients shows a 49% complete response rate at 6 months anda 30% complete response rate at 12 months. BCG refractory patientsshowed a 56% complete response rate at 6 months and a 44% completeresponse rate at 12 months. Finally, BCG relapsing patients showed a 35%response rate at 6 months and an 18% response rate at 12 months. Thisdemonstrates that CG0070 is effective for treating BCG unresponsive, BCGrelapsing, and BCG refractory patients.

TABLE 6 Cancer Stage of Patients Evaluated Cancer Stage CIS alone(N =31) 46%  CIS + Ta (N = 11) 16%  CIS + T1 (N = 4) 6% CIS Ta T1 (N = 2) 3%Ta (N = 11) 16%  T1 (N = 6) 9% Ta, T1 (N = 2) 3%

TABLE 7 Response Rate after 6 and 12 months # CR @ 6 CR @ 6 # CR @ 12 CR@ 12 N Proportion Months Months Months Months BCG Unresponsive (0-6,6-12) 43 64% 21 49% 13 30% BCG Refractory (0-6) 27 40% 15 56% 12 44% BCGRelapsing (6-12, 12+) 34 51% 12 35% 6 18% Insufficient Data 6  9% 2 33%0  0%

Responsiveness of patients with BCG unresponsive bladder cancer was alsoanalyzed by cancer subtype, as summarized in Table 8. As shown in Table8, patients with papillary cancers showed a somewhat higher completeresponse rate than patients with CIS-containing cancers (62% vs 46% CRat 6 months and 55% vs 26% CR at 12 months).

TABLE 8 Complete Response Rates in BCG Unresponsive Sub Populations. 6Month 12 Month # CR @ 6 CR @ 6 # CR @ 12 CR @ 12 Months Months MonthsMonths CIS alone 8 53% 3 21% CIS + Ta 4 50% 4 50% CIS + T1 0  0% 0  0%CIS + Ta + T1 1 50% 0  0% Total Cis-containing 13 46% 7 26% Ta 4 67% 250% T1 4 80% 4 80% Ta + T1 0  0% 0  0% Total papillary 8 73% 6 75% TotalBCG 21 54% 13 36% Unresponsive

The association between time to relapse and responsiveness to CG0700therapy was also evaluated. Out of 14 counted subjects that relapsedafter achieving a CR, 64% ( 9/14) relapsed at 6-month after achieving CRat 3-month. CR is sustainable starting at 9-month if relapse does notoccur after this timepoint. No Ta/T1 patients relapsed after achievingCR.

In summary, this phase II study of CG0700 demonstrates its effectivenessfor treating both CIS-containing and papillary bladder cancer. Patientswith unresponsive, BCG refractory, and BCG relapsing bladder cancer allshowed high response rates to CG0700 demonstrating its efficacy in theseunderserved patient groups. The high level of efficacy of the CG0700therapy suggests that it could be a viable alternative to radicalcystectomy, allowing patients with non-muscle invasive bladder cancer topreserve their bladders and increasing the quality of life for thesepatients.

Example 2: Phase III Clinical Trial of CG0070 for Treatment of Patientswith Bladder Cancer

This study shows the effect of a treatment regimen with CG0070comprising an induction phase and a maintenance phase. Patients withbladder cancer are administered C00700 therapy once per week for sixweeks at month 0 during an induction phase. At month three, patients arereassessed. Patients with persistent disease will continue the inductiontherapy and receive a weekly CG0070 for six weeks at three months.Patients who show a complete response will begin a maintenance phase andreceive weekly CG0070 for three weeks at three months. At month 6, allpatients will receive CG0070 weekly for three weeks in a maintenancephase. At months 12 and 18, patients will receive CG0070 weekly forthree weeks during the maintenance phase. Patients will receive amaximum of 21 doses of CG0700. Complete response rate is assessed.

Example 3: Treatment of Patients with Ta or T1 Non-Muscle InvasiveBladder Cancer Who Have Not Received TURBT

This study shows the effect of treatment of patients with Ta or T1non-muscle invasive bladder cancer who have not received atransurothelial resection of bladder tumor (TURBT), for example in aneoadjuvant setting. Patients who have not received a TURBT areadministered CG0070. Patients include those who are ineligible for TURBTand patients who are considered to have non-resectable Ta or T1 stagebladder cancer. Complete response rate is assessed.

What is claimed is:
 1. A method of treating bladder cancer in anindividual or preserving bladder in an individual, comprisingintravesically administering to the individual an effective amount of anoncolytic virus once per week for six weeks during an induction phase,and subsequently intravesically administering to the individual aneffective amount of the oncolytic virus once per week for three weeksevery three or six months during a maintenance phase, wherein the startof the induction phase and the start of the maintenance phase areseparated by about three months or about six months, and wherein theoncolytic virus comprises a viral vector comprising a tumorcell-specific promoter operably linked to a viral gene essential forreplication of the oncolytic virus, and a heterologous gene encoding animmune-related molecule.
 2. The method of claim 1, wherein the start ofthe induction phase and the start of the maintenance phase are separatedby about three months.
 3. The method of claim 1, wherein the start ofthe induction phase and the start of the maintenance phase are separatedby about six months.
 4. The method of claim 1, wherein the inductionphase comprises administering to the individual an effective amount ofan oncolytic virus once per week for six weeks on month zero and monththree of a treatment regimen.
 5. The method of claim 1, wherein theindividual has carcinoma in situ.
 6. The method of claim 5, wherein theindividual does not have a concurrent papillary carcinoma of Ta or T1stage.
 7. The method of claim 1, wherein the individual has Ta or T1stage bladder cancer.
 8. The method of claim 1, wherein the individualis unresponsive to BCG treatment or has disease recurrence subsequent toBCG treatment.
 9. The method of claim 8, wherein the individual hasfailed the BCG treatment within about 12 months after the lastadministration of BCG.
 10. The method of claim 1, wherein the individualhas not received a cystectomy.
 11. The method of claim 10, wherein theindividual has refused or is ineligible for a cystectomy.
 12. The methodof claim 1, wherein the tumor cell-specific promoter is an E2F-1promoter.
 13. The method of claim 1, wherein the immune-related moleculeis GM-CSF.
 14. The method of claim 1, wherein the oncolytic virus is anoncolytic adenovirus.
 15. The method of claim 1, wherein the oncolyticvirus is an adenovirus serotype 5, wherein the endogenous E1a promoterof the native adenovirus serotype 5 is replaced by the human E2F-1promoter, and the endogenous E3 19kD coding region of the nativeadenovirus serotype 5 is replaced by a nucleic acid encoding humanGM-CSF.
 16. The method of claim 15, wherein the oncolytic virus isCG0070.
 17. The method of claim 1, wherein the oncolytic virus isadministered at a dose of about 1×10⁸ to about 1×10¹⁴ viral particles.18. The method of claim 1, further comprising intravesicallyadministering to the individual a transduction enhancing agent prior tothe administration of the oncolytic virus.
 19. The method of claim 18,wherein the transduction enhancing agent is N-Dodecyl-β-D-maltoside(DDM).
 20. The method of claim 1, wherein the oncolytic virus isadministered as a single therapeutic agent.
 21. The method of claim 1,wherein the individual is human.
 22. The method of claim 1, wherein theindividual is administered the oncolytic virus once per week for sixweeks on month zero during the induction phase and is reevaluated atmonth three.
 23. The method of claim 22, wherein the individual beginsthe maintenance phase at month three.
 24. The method of claim 23,wherein the maintenance phase comprises administering the oncolyticvirus once per week for three weeks every three months for nine monthsand subsequently administering the oncolytic virus once per week forthree weeks every six months.
 25. The method of claim 22, wherein theindividual receives a second induction dose of oncolytic virus once perweek for six weeks at month three.
 26. The method of claim 25, whereinthe maintenance phase comprises administering the oncolytic virus onceper week for three weeks every three months for six months andsubsequently administering the oncolytic virus once per week for threeweeks every six months.
 27. The method of claim 1, wherein themaintenance phase comprises: (i) administering the oncolytic virus onceper week for three weeks every three months for six to nine months, andsubsequently (ii) administering the oncolytic virus once per week forthree weeks every six months.
 28. The method of claim 1, wherein theindividual has non-muscle-invasive bladder cancer.
 29. The method ofclaim 7, wherein the individual does not have carcinoma in situ.